Cyclic AMP-specific phosphodiesterase inhibitors

ABSTRACT

Pyrrole compounds that are potent and selective inhibitors of PDE4, as well as methods of making the same, are disclosed. Use of the compounds in the treatment of inflammatory diseases and other diseases involving elevated levels of cytokines, as well as central nervous system (CNS) disorders, also is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of provisional application SerialNo. 60/171,954, filed Dec. 23, 1999.

FIELD OF INVENTION

The present invention relates to a series of compounds that are potentand selective inhibitors of cyclic adenosine 3′,5′-monophosphatespecific phosphodiesterase (cAMP-specific PDE). In particular, thepresent invention relates to a series of novel pyrrole compounds thatare useful for inhibiting the function of cAMP-specific PDE, inparticular, PDE4, as well as methods of making the same, pharmaceuticalcompositions containing the same, and their use as therapeutic agents,for example, in treating inflammatory diseases and other diseasesinvolving elevated levels of cytokines and proinflammatory mediators.

BACKGROUND OF THE INVENTION

Chronic inflammation is a multi-factorial disease complicationcharacterized by activation of multiple types of inflammatory cells,particularly cells of lymphoid lineage (including T lymphocytes) andmyeloid lineage (including granulocytes, macrophages, and monocytes).Proinflammatory mediators, including cytokines, such as tumor necrosisfactor (TNF) and interleukin-1 (IL-1), are produced by these activatedcells. Accordingly, an agent that suppresses the activation of thesecells, or their production of proinflammatory cytokines, would be usefulin the therapeutic treatment of inflammatory diseases and other diseasesinvolving elevated levels of cytokines.

Cyclic adenosine monophosphate (cAMP) is a second messenger thatmediates the biologic responses of cells to a wide range ofextracellular stimuli. When the appropriate agonist binds to specificcell surface receptors, adenylate cyclase is activated to convertadenosine triphosphate (ATP) to cAMP. It is theorized that the agonistinduced actions of cAMP within the cell are mediated predominately bythe action of cAMP-dependent protein kinases. The intracellular actionsof cAMP are terminated by either a transport of the nucleotide to theoutside of the cell, or by enzymatic cleavage by cyclic nucleotidephosphodiesterases (PDEs), which hydrolyze the 3′-phosphodiester bond toform 5′-adenosine monophosphate (5′-AMP). 5′-AMP is an inactivemetabolite. The structures of cAMP and 5′-AMP are illustrated below.

Elevated levels of cAMP in human myeloid and lymphoid lineage cells areassociated with the suppression of cell activation. The intracellularenzyme family of PDEs, therefore, regulates the level of cAMP in cells.PDE4 is a predominant PDE isotype in these cells, and is a majorcontributor to cAMP degradation. Accordingly, the inhibition of PDEfunction would prevent the conversion of cAMP to the inactive metabolite51-AMP and, consequently, maintain higher cAMP levels, and, accordingly,suppress cell activation (see Beavo et al., “Cyclic NucleotidePhosphodiesterases: Structure, Regulation and Drug Action,” Wiley andSons, Chichester, pp. 3-14, (1990)); Torphy et al., Drug News andPerspectives, 6, pp. 203-214 (1993); Giembycz et al., Clin. Exp.Allergy, 22, pp. 337-344 (1992)).

In particular, PDE4 inhibitors, such as rolipram, have been shown toinhibit production of TNFα and partially inhibit IL-1β release bymonocytes (see Semmler et al., Int. J. Immunopharmacol., 15, pp.409-413, (1993); Molnar-Kimber et al., Mediators of Inflammation, 1, pp.411-417, (1992)). PDE4 inhibitors also have been shown to inhibit theproduction of superoxide radicals from human polymorphonuclearleukocytes (see Verghese et al., J. Mol. Cell. Cardiol., 21 (Suppl. 2),S61 (1989); Nielson et al., J. Allergy Immunol., 86, pp. 801-808,(1990)); to inhibit the release of vasoactive amines and prostanoidsfrom human basophils (see Peachell et al., J. Immunol, 148, pp.2503-2510, (1992)); to inhibit respiratory bursts in eosinophils (seeDent et al., J. Pharmacol., 103, pp. 1339-1346, (1991)); and to inhibitthe activation of human T-lymphocytes (see Robicsek et al., Biochem.Pharmacol., 42, pp. 869-877, (1991)).

Inflammatory cell activation and excessive or unregulated cytokine(e.g., TNFα and IL-1β) production are implicated in allergic,autoimmune, and inflammatory diseases and disorders, such as rheumatoidarthritis, osteoarthritis, gouty arthritis, spondylitis, thyroidassociated ophthalmopathy, Behcet's disease, sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shocksyndrome, asthma, chronic bronchitis, adult respiratory distresssyndrome, chronic pulmonary inflammatory disease, such as chronicobstructive pulmonary disease, silicosis, pulmonary sarcoidosis,reperfusion injury of the myocardium, brain, and extremities, fibrosis,cystic fibrosis, keloid formation, scar formation, atherosclerosis,transplant rejection disorders, such as graft vs. host reaction andallograft rejection, chronic glomerulonephritis, lupus, inflammatorybowel disease, such as Crohn's disease and ulcerative colitis,proliferative lymphocyte diseases, such as leukemia, and inflammatorydermatoses, such as atopic dermatitis, psoriasis, and urticaria.

Other conditions characterized by elevated cytokine levels include braininjury due to moderate trauma (see Dhillon et al., J. Neurotrauma, 12,pp. 1035-1043 (1995); Suttorp et al., J. Clin. Invest., 91, pp.1421-1428 (1993)), cardiomyopathies, such as congestive heart failure(see Bristow et al., Circulation, 97, pp. 1340-1341 (1998)), cachexia,cachexia secondary to infection or malignancy, cachexia secondary toacquired immune deficiency syndrome (AIDS), ARC (AIDS related complex),fever myalgias due to infection, cerebral malaria, osteoporosis and boneresorption diseases, keloid formation, scar tissue formation, andpyrexia.

In particular, TNFα has been identified as having a role with respect tohuman acquired immune deficiency syndrome (AIDS). AIDS results from theinfection of T-lymphocytes with Human Immunodeficiency Virus (HIV).Although HIV also infects and is maintained in myeloid lineage cells,TNF has been shown to upregulate HIV infection in T-lymphocytic andmonocytic cells (see Poli et al., Proc. Natl. Acad. Sci. USA, 87, pp.782-785, (1990)).

Several properties of TNFα, such as stimulation of collagenases,stimulation of angiogenesis in vivo, stimulation of bone resorption, andan ability to increase the adherence of tumor cells to endothelium, areconsistent with a role for TNF in the development and metastatic spreadof cancer in the host. TNFα recently has been directly implicated in thepromotion of growth and metastasis of tumor cells (see Orosz et al., J.Exp. Med., 177, pp. 1391-1398, (1993)).

PDE4 has a wide tissue distribution. There are at least four genes forPDE4 of which multiple transcripts from any given gene can yield severaldifferent proteins that share identical catalytic sites. The amino acididentity between the four possible catalytic sites is greater than 85%.Their shared sensitivity to inhibitors and their kinetic similarityreflect the functional aspect of this level of amino acid identity. Itis theorized that the role of these alternatively expressed PDE4proteins allows a mechanism by which a cell can differentially localizethese enzymes intracellularly and/or regulate the catalytic efficiencyvia post translational modification. Any given cell type that expressesthe PDE4 enzyme typically expresses more than one of the four possiblegenes encoding these proteins.

Investigators have shown considerable interest in the use of PDE4inhibitors as anti-inflammatory agents. Early evidence indicates thatPDE4 inhibition has beneficial effects on a variety of inflammatorycells such as monocytes, macrophages, T-cells of the Th-1 lineage, andgranulocytes. The synthesis and/or release of many pro-inflammatorymediators, such as cytokines, lipid mediators, superoxide, and biogenicamines, such as histamine, have been attenuated in these cells by theaction of PDE4 inhibitors. The PDE4 inhibitors also affect-othercellular functions including T-cell proliferation, granulocytetransmigration in response to chemotoxic substances, and integrity ofendothelial cell junctions within the vasculature.

The design, synthesis, and screening of various PDE4 inhibitors havebeen reported. Methyl-xanthines, such as caffeine and theophylline, werethe first PDE inhibitors discovered, but these compounds arenonselective with respect to which PDE is inhibited. The drug rolipram,an antidepressant agent, was one of the first reported specific PDE4inhibitors. Rolipram, having the following structural formula, has areported 50% Inhibitory Concentration (IC₅₀) of about 200 nM (nanomolar)with respect to inhibiting recombinant human PDE4.

Investigators have continued to search for PDE4 inhibitors that are moreselective with respect to inhibiting PDE4, that have a lower IC₅₀ thanrolipram, and that avoid the undesirable central nervous system (CNS)side effects, such as retching, vomiting, and sedation, associated withthe administration of rolipram. One class of compounds is disclosed inFeldman et al. U.S. Pat. No. 5,665,754. The compounds disclosed thereinare substituted pyrrolidines having a structure similar to rolipram. Oneparticular compound, having the following structural formula, has anIC₅₀ with respect to human recombinant PDE4 of about 2 nM. Inasmuch as afavorable separation of emetic side effect from efficacy was observed,these compounds did not exhibit a reduction in undesirable CNS effects.

In addition, several companies are now undertaking clinical trials ofother PDE4 inhibitors. However, problems relating to efficacy andadverse side effects, such as emesis and central nervous systemdisturbances, remain unsolved.

Accordingly, compounds that selectively inhibit PDE4, and that reduce oreliminate the adverse CNS side effects associated with prior PDE4inhibitors, would be useful in the treatment of allergic andinflammatory diseases, and other diseases associated with excessive orunregulated production of cytokines, such as TNF. In addition, selectivePDE4 inhibitors would be useful in the treatment of diseases that areassociated with elevated cAMP levels or PDE4 function in a particulartarget tissue.

SUMMARY OF THE INVENTION

The present invention is directed to potent and selective PDE4inhibitors useful in treatment of diseases and conditions whereinhibition of PDE4 activity is considered beneficial. The present PDE4inhibitors unexpectedly reduce or eliminate the adverse CNS side effectsassociated with prior PDE4 inhibitors.

In particular, the present invention is directed to pyrrole compoundshaving the structural formula (I) or (II):

wherein R¹ is selected from the group consisting of optionallysubstituted aryl, alkyl, cycloalkyl, heteroaryl, alkaryl, aralkyl,heteroaralkyl, and heteroalkaryl;

R² is alkyl or hydrogen;

R³ is selected from the group consisting of alkyl, alkoxy, alkoxyalkyl,hydroxyl, aryl, heteroaryl, aryloxyalkyl, aralkoxyalkyl, C(═O)alkyl,NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H, OC(═O)alkyl, nitro, halo,alkylthio, SO₂(alkyl), SO₃H, and haloalkyl;

R⁴ and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic or heterocyclic ring;and

n is 0 through 3.

The present invention also is directed to pharmaceutical compositionscontaining one or more of the compounds of structural formula (I) or(II), to use of the compounds and compositions containing the compoundsin the treatment of a disease or disorder, and to methods of preparingthe compounds and intermediates involved in the synthesis of thecompounds of structural formula (I) and (II).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains plots of TNFα concentration in serum (pg/mL) vs.concentration of PDE4 inhibitors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to compounds having a structuralformula (I) or (II):

wherein

R¹ is selected from the group consisting of optionally substitutedalkyl, cycloalkyl, aryl, heteroaryl, alkaryl, aralkyl, heteroaralkyl,and heteroalkaryl;

R² is alkyl or hydrogen;

R³ is selected from the group consisting of alkyl, alkoxy, alkoxyalkyl,hydroxy, aryl, heteroaryl, aryloxyalkyl, aralkoxyalkyl, C(═O)alkyl,NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H, OC(═O)alkyl, nitro, halo,alkylthio, SO₂(alkyl), SO₃H, and haloalkyl;

R⁴ and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic or heterocyclic ring;and

n is 0 through 3.

As used herein, the term “alkyl,” alone or in combination, is defined toinclude straight chain and branched chain, and bridged, saturatedhydrocarbon groups containing one to 16 carbon atoms. The term “loweralkyl” is defined herein as an alkyl group having one through six carbonatoms (C₁-C₆) Examples of lower alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl,neopentyl, -hexyl, and the like.

The term “bridged alkyl” is defined herein as a C₆-C₁₆ bicyclic orpolycyclic hydrocarbon group, for example, norboryl, adamantyl,bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, ordecahydronaphthyl.

The term “cycloalkyl” is defined herein to include cyclic C₃-C₇hydrocarbon groups. Examples of cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

The term “haloalkyl” is defined herein as an alkyl group substitutedwith one or more halo substituents, either fluoro, chloro, bromo, iodo,or combinations thereof. Similarly, “halocycloalkyl” is defined as acycloalkyl group having one or more halo substituents.

The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl, that can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents selected from halo, alkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino,alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.Exemplary aryl groups include phenyl, naphthyl, tetrahydronaphthyl,2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl,4-methoxyphenyl, 3-trifluoromethylphenyl, 4-nitrophenyl, and the like.

The term “heteroaryl” is defined herein as a monocyclic or bicyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring, and which can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents, like halo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl,isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

The term “aralkyl” is defined herein as a previously defined alkylgroup, wherein one of the hydrogen atoms is replaced by an aryl group asdefined herein, for example, a phenyl group optionally having one ormore substituents, for example, halo, alkyl, alkoxy, and the like. Anexample of an aralkyl group is a benzyl group.

The term “alkaryl” is defined herein as a previously defined aryl group,wherein one of the hydrogen atoms is replaced by an alkyl, cycloalkyl,haloalkyl, or halocycloalkyl group.

The term “heteroaralkyl” and “heteroalkaryl” are defined similarly asthe terms “aralkyl” and “alkaryl,” however, the aryl group is replacedby a heteroaryl group as previously defined.

The term “heterocycle” or “heterocyclic ring” is defined as a 5- or6-membered nonaromatic ring having one or more heteroatoms selected fromoxygen, nitrogen, and sulfur present in the ring. Nonlimiting examplesinclude tetrahydrofuran, piperidine, piperazine, sulfolane, morpholine,tetrahydropyran, dioxane, and the like. A “carbocyclic ring” issimilarly defined, but the ring contains solely carbon atoms.

The term “halogen” or “halo” is defined herein to include fluorine,chlorine, bromine, and iodine.

The terms “alkoxy,” “aryloxy,” and “aralkoxy” are defined as —OR,wherein R is alkyl, aryl, and aralkyl, respectively.

The term “alkoxyalkyl” is defined as an alkoxy group appended to analkyl group. The terms “aryloxyalkyl” and “aralkoxyalkyl” are similarlydefined as an aryloxy or aralkoxy group appended to an alkyl group.

The term “hydroxy” is defined as —OH.

The term “hydroxyalkyl” is defined as a hydroxy group appended to analkyl group.

The term “amino” is defined as —NH₂.

The term “alkylamino” is defined as —NR₂ wherein at least one R is alkyland the second R is alkyl or hydrogen.

The term “acylamino” is defined as RC(═O)NH, wherein R is alkyl or aryl.

The term “nitro” is defined as —NO₂.

The term “alkylthio” is defined as —SR, where R is alkyl.

The term “alkylsulfinyl” is defined as R—S(O)₂, where R is alkyl.

The term “alkylsulfonyl” is defined as R—S(O₃), where R is alkyl.

In preferred embodiments, R¹ is selected from the group consisting ofaryl, heteroaryl, alkaryl, aralkyl, and cycloalkyl; R² is selected fromthe group consisting of hydrogen and lower alkyl.

In most preferred embodiments, R¹ is aryl, heteroaryl, cycloalkyl, oraralkyl, optionally substituted with one or more of NR⁴R⁵, halo, alkyl,C(═O)alkyl, SO₂(alkyl), C(═O)NR⁴R⁵, SO₃H, NO₂, CO₂H, C(═O)Oalkyl, andtrifluoromethyl; R² is hydrogen or C₁₋₂alkyl, and R³ is hydrogen. R¹typically is phenyl or pyridyl, optionally substituted, and R² ishydrogen or ethyl.

The present invention includes all possible stereoisomers and geometricisomers of compounds of structural formulae (I) and (II), and includesnot only racemic compounds but also the optically active isomers aswell. When a compound of structural formulae (I) and (II) is desired asa single enantiomer, it can be obtained either by resolution of thefinal product or by stereospecific synthesis from either isomericallypure starting material or use of a chiral auxiliary reagent, forexample, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-888(1997). Resolution of the final product, an intermediate, or a startingmaterial can be achieved by any suitable method known in the art.Additionally, in situations where tautomers of the compounds ofstructural formulae (I) and (II) are possible, the present invention isintended to include all tautomeric forms of the compounds. Asdemonstrated hereafter, specific stereoisomers exhibit an exceptionalability to inhibit PDE4 without manifesting the adverse CNS side effectstypically associated with PDE4 inhibitors.

Compounds of structural formula (I) and (II) which contain acidicmoieties can form pharmaceutically acceptable salts with suitablecations. Suitable pharmaceutically acceptable cations include alkalimetal (e.g., sodium or potassium) and alkaline earth metal (e.g.,calcium or magnesium) cations. The pharmaceutically acceptable salts ofthe compounds of structural formula (I) and (II), which contain a basiccenter, are acid addition salts formed with pharmaceutically acceptableacids. Examples include the hydrochloride, hydrobromide, sulfate orbisulfate, phosphate or hydrogen phosphate, acetate, benzoate,succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate,methanesulfonate, benzenesulphonate, and p-toluenesulphonate salts. Inlight of the foregoing, any reference to compounds of the presentinvention appearing herein is intended to include compounds ofstructural formula (I) and (II), as well as pharmaceutically acceptablesalts and solvates thereof.

The compounds of the present invention can be therapeuticallyadministered as the neat chemical, but it is preferable to administercompounds of structural formula (I) and (II) as a pharmaceuticalcomposition or formulation. Accordingly, the present invention furtherprovides for pharmaceutical formulations comprising a compound ofstructural formula (I) and/or (II), or pharmaceutically acceptable saltsthereof, together with one or more pharmaceutically acceptable carriersand, optionally, other therapeutic and/or prophylactic ingredients. Thecarriers are “acceptable” in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

In particular, a selective PDE4 inhibitor of the present invention isuseful alone or in combination with a second antiinflammatorytherapeutic agent, for example, a therapeutic agent targeting TNFα, suchas ENBREL® or REMICADE®, which have utility in treating rheumatoidarthritis. Likewise, therapeutic utility of IL-1 antagonism has alsobeen shown in animal models for rheumatoid arthritis. Thus, it isenvisioned that IL-1 antagonism, in combination with PDE4 inhibition,which attenuates TNFα, would be efficacious.

The present PDE4 inhibitors are useful in the treatment of a variety ofallergic, autoimmune, and inflammatory diseases.

The term “treatment” includes preventing, lowering, stopping, orreversing the progression of severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

In particular, inflammation is a localized, protective response elicitedby injury or destruction of tissues, which serves to destroy, dilute orwall off (i.e., sequester) both the injurious agent and the injuredtissue. The term “inflammatory disease,” as used herein, means anydisease in which an excessive or unregulated inflammatory response leadsto excessive inflammatory symptoms, host tissue damage, or loss oftissue function. Additionally, the term “autoimmune disease,” as usedherein, means any group of disorders in which tissue injury isassociated with humoral or cell-mediated responses to the body'sownconstituents The term “allergic disease,” as used herein, means anysymptoms, tissue damage, or loss of tissue function resulting fromallergy. The term “arthritic disease,” as used herein, means any of alarge family of diseases that are characterized by inflammatory lesionsof the joints attributable to a variety of etiologies. The term“dermatitis,” as used herein, means any of a large family of diseases ofthe skin that are characterized by inflammation of the skin attributableto a variety of etiologies. The term “transplant rejection,” as usedherein, means any immune reaction directed against grafted tissue(including organ and cell (e.g., bone marrow)), characterized by a lossof function of the grafted and surrounding tissues, pain, swelling,leukocytosis and thrombocytopenia.

The present invention also provides a method of modulating cAMP levelsin a mammal, as well as a method of treating diseases characterized byelevated cytokine levels.

The term “cytokine,” as used herein, means any secreted polypeptide thataffects the functions of other cells, and that modulates interactionsbetween cells in the immune or inflammatory response. Cytokines include,but are not limited to monokines, lymphokines, and chemokines regardlessof which cells produce them. For instance, a monokine is generallyreferred to as being produced and secreted by a monocyte, however, manyother cells produce monokines, such as natural killer cells,fibroblasts, basophils, neutrophils, endothelial cells, brainastrocytes, bone marrow stromal cells, epidermal keratinocytes, andB-lymphocytes. Lymphokines are generally referred to as being producedby lymphocyte cells. Examples of cytokines include, but are not limitedto, interleukin-1 (IL-1), interleukin-6 (IL-6), Tumor Necrosis Factoralpha (TNFα), and Tumor Necrosis Factor beta (TNFβ).

The present invention further provides a method of reducing TNF levelsin a mammal, which comprises administering an effective amount of acompound of structural formula (II) to the mammal. The term “reducingTNF levels,” as used herein, means either:

a) decreasing excessive in vivo TNF levels in a mammal to normal levelsor below normal levels by inhibition of the in viva release of TNF byall cells, including but not limited to monocytes or macrophages; or

b) inducing a down-regulation, at the translational or transcriptionlevel, of excessive in vivo TNF levels in a mammal to normal levels orbelow normal levels; or

c) inducing a down-regulation, by inhibition of the direct synthesis ofTNF as a postranslational event.

Moreover, the compounds of the present invention are useful insuppressing inflammatory cell activation. The term “inflammatory cellactivation,” as used herein, means the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes, polymorphonuclearleukocytes, mast cells, basophils, eosinophils, dendritic cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

The compounds of the present invention also are useful in causing airwaysmooth muscle relaxation, bronchodilation, and prevention ofbronchoconstriction.

The compounds of the present invention, therefore, are useful intreating such diseases as arthritic diseases (such as rheumatoidarthritis), osteoarthritis, gouty arthritis, spondylitis,thyroid-associated ophthalmopathy, Behcet disease, sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shocksyndrome, asthma, chronic bronchitis, allergic rhinitis, allergicconjunctivitis, vernal conjunctivitis, eosinophilic granuloma, adult(acute) respiratory distress syndrome (ARDS), chronic pulmonaryinflammatory disease (such as chronic obstructive pulmonary disease),silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium,brain or extremities, brain or spinal cord injury due to minor trauma,fibrosis including cystic fibrosis, keloid formation, scar tissueformation, atherosclerosis, autoimmune diseases, such as systemic lupuserythematosus (SLE) and transplant rejection disorders (e.g., graft vs.host (GvH) reaction and allograft rejection), chronicglomerulonephritis, inflammatory bowel diseases, such as Crohn's diseaseand ulcerative colitis, proliferative lymphocytic diseases, such asleukemias (e.g. chronic lymphocytic leukemia; CLL) (see Mentz et al.,Blood 88, pp. 2172-2182 (1996)), and inflammatory dermatoses, such asatopic dermatitis, psoriasis, or urticaria.

Other examples of such diseases or related conditions includecardiomyopathies, such as congestive heart failure, pyrexia, cachexia,cachexia secondary to infection or malignancy, cachexia secondary toacquired immune deficiency syndrome (AIDS), ARC (AIDS-related complex),cerebral malaria, osteoporosis and bone resorption diseases, and feverand myalgias due to infection. In addition, the compounds of the presentinvention are useful in the treatment of diabetes insipidus and centralnervous system disorders, such as depression and multiinfarct dementia.

Compounds of the present invention also have utility outside of thattypically known as therapeutic. For example, the present compounds canfunction as organ transplant preservatives (see Pinsky et al., J. Clin.Invest., 92, pp. 2994-3002 (1993)) as well.

Selective PDE4 inhibitors also can be useful in the treatment ofdiabetes insipidus (Kidney Int., 37, p. 362, (1990); Kidney Int., 35, p.494, (1989)) and central nervous system disorders, such as multiinfarctdementia (Nicholson, Psychopharmacology, 101, p. 147 (1990)), depression(Eckman et al., Curr. Ther. Res., 43, p. 291 (1988)), anxiety and stressresponses (Neuropharmacology, 38, p. 1831 (1991)), cerebral ischemia(Eur. J. Pharmacol., 272, p. 107 (1995)), tardive dyskinesia (J. Clin.Pharmocol., 16, p. 304 (1976)), Parkinson's disease (see Neurology, 25,p. 722 (1975); Clin. Exp. Pharmacol, Physiol., 26, p. 421 (1999)), andpremenstrual syndrome. With respect to depression, PDE4-selectiveinhibitors show efficacy in a variety of animal models of depressionsuch as the “behavioral despair” or Porsolt tests (Eur. J. Pharmacol.,47, p. 379 (1978); Eur. J. Pharmacol., 57, p. 431 (1979);Antidepressants: neurochemical, behavioral and clinical prospectives,Enna, Malick, and Richelson, eds., Raven Press, p. 121 (1981)), and the“tail suspension test” (Psychopharmacology, 85, p. 367 (1985)). Recentresearch findings show that chronic in vivo treatment by a variety ofantidepressants increase the brain-derived expression of PDE4 (J.Neuroscience, 19, p. 610 (1999)). Therefore, a selective PDE4 inhibitorcan be used alone or in conjunction with a second therapeutic agent in atreatment for the four major classes of antidepressants:electroconvulsive procedures, monoamine oxidase inhibitors, andselective reuptake inhibitors of serotonin or norepinephrine. SelectivePDE4 inhibitors also can be useful in applications that modulatebronchodilatory activity via direct action on bronchial smooth musclecells for the treatment of asthma.

Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amounteffective to prevent development of, or to alleviate the existingsymptoms of, the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

A “therapeutically effective dose” refers to that amount of the compoundthat results in achieving the desired effect. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which is expressed as the ratio between LD₅₀ and ED₅₀. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom such data can be used in formulating a dosage range for use inhumans. The dosage of such compounds preferably lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed, and the route of administration utilized.

The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active moiety which are sufficient to maintain thetherapeutic effects.

As appreciated by persons skilled in the art, reference herein totreatment extends to prophylaxis, as well as to treatment of establisheddiseases or symptoms. It is further appreciated that the amount of acompound of the invention required for use in treatment varies with thenature of the condition being treated, and with the age and thecondition of the patient, and is ultimately determined by the attendantphysician or veterinarian. In general, however, doses employed for adulthuman treatment typically are in the range of 0.001 mg/kg to about 100mg/kg per day. The desired dose can be conveniently administered in asingle dose, or as multiple doses administered at appropriate intervals,for example as two, three, four or more subdoses per day. In practice,the physician determines the actual dosing regimen which is mostsuitable for an individual patient, and the dosage varies with the age,weight, and response of the particular patient. The above dosages areexemplary of the average case, but there can be individual instances inwhich higher or lower dosages are merited, and such are within the scopeof the present invention.

Formulations of the present invention can be administered in a standardmanner for the treatment of the indicated diseases, such as orally,parenterally, transmucosally (e.g., sublingually or via buccaladministration), topically, transdermally, rectally, via inhalation(e.g., nasal or deep lung inhalation). Parenteral administrationincludes, but is not limited to intravenous, intraarterial,intraperitoneal, subcutaneous, intramuscular, intrathecal, andintraarticular. Parenteral administration also can be accomplished usinga high pressure technique, like POWDERJECT™.

For buccal administration, the composition can be in the form of tabletsor lozenges formulated in conventional manner. For example, tablets andcapsules for oral administration can contain conventional excipientssuch as binding agents (for example, syrup, accacia, gelatin, sorbitol,tragacanth, mucilage of starch or polyvinylpyrrolidone), fillers (forexample, lactose, sugar, microcrystalline, cellulose, maize-starch,calcium phosphate or sorbitol), lubricants (for example, magnesium,stearate, stearic acid, talc, polyethylene glycol or silica),disintegrants (for example, potato starch or sodium starch glycollate),or wetting agents (for example, sodium lauryl sulfate). The tablets canbe coated according to methods well known in the art.

Alternatively, the compounds of the present invention can beincorporated into oral liquid preparations such as aqueous or oilysuspensions, solutions, emulsions, syrups, or elixirs, for example.Moreover, formulations containing these compounds can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can contain conventional additives, suchas suspending agents, such as sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxyethylcellulose,hydroxyropylmethylcellulose, carboxymethyl cellulose, aluminum stearategel, and hydrogenated edible fats; emulsifying agents, such as lecithin,sorbitan monooleate, or acacia; nonaqueous vehicles (which can includeedible oils), such as almond oil, fractionated coconut oil, oily esters,propylene glycol, and ethyl alcohol; and preservatives, such as methylor propyl p-hydroxybenzoate and sorbic acid.

Such preparations also can be formulated as suppositories, e.g.,containing conventional suppository bases, such as cocoa butter or otherglycerides. Compositions for inhalation typically can be provided in theform of a solution, suspension, or emulsion that can be administered asa dry powder or in the form of an aerosol using a conventionalpropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Typical topical and transdermal formulations comprise conventionalaqueous or nonaqueous vehicles, such as eye drops, creams, ointments,lotions, and pastes, or are in the form of a medicated plaster, patch,or membrane.

Additionally, compositions of the present invention can be formulatedfor parenteral administration by injection or continuous infusion.Formulations for injection can be in the form of suspensions, solutions,or emulsions in oily or aqueous vehicles, and can contain formulationagents, such as suspending, stabilizing, and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water)before use.

A composition in accordance with the present invention also can beformulated as a depot preparation. Such long acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Accordingly, thecompounds of the invention can be formulated with suitable polymeric orhydrophobic materials (e.g., an emulsion in an acceptable oil), ionexchange resins, or as sparingly soluble derivatives (e.g., a sparinglysoluble salt).

For veterinary use, a compound of formula (I) or (II), or nontoxic saltsthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (I) or (II), togetherwith a pharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition comprising a compound of formula (I) or (II),which process comprises mixing a compound of formula (I) or (II),together with a pharmaceutically acceptable diluent or carrier therefor.

Specific, nonlimiting examples of compounds of structural formula (I)and (II) are provided below, the synthesis of which were performed inaccordance with the procedures set forth below.

Generally, compounds of structural formula (I) and (II) can be preparedaccording to the following synthetic scheme. In the scheme describedbelow, it is understood in the art that protecting groups can beemployed where necessary in accordance with general principles ofsynthetic chemistry. These protecting groups are removed in the finalsteps of the synthesis under basic, acidic, or hydrogenolytic conditionswhich are readily apparent to those skilled in the art. By employingappropriate manipulation and protection of any chemical functionalities,synthesis of compounds of structural formula (I) and (II) notspecifically set forth herein can be accomplished by methods analogousto the schemes set forth below.

Unless otherwise noted, all starting materials were obtained fromcommercial suppliers and used without further purification. Allreactions and chromatography fractions were analyzed by thin-layerchromatography on 250-mm silica gel plates, visualized with UV(ultraviolet) light and I₂ (iodine) stain. Flash column chromatographywas carried out using Biotage 40M silica gel (230-400 mesh). Productsand intermediates were purified by flash chromatography or reverse-phaseHPLC.

As illustrated below, the compounds of general structural formula (I)are prepared by reacting a compound having the formula,

with sodium metal to generate a carbanion, then reacting the carbanionwith a compound having the formula

wherein Hal is a halo, typically bromo, to generate an intermediatecompound having the formula

This intermediate compound then is reacted with a primary amine havingthe formula R¹NH₂ to provide a compound of structural formula (I).

A compound of structural formula (II) is prepared by reacting a compoundhaving the formula

with chloroacetone to generate an intermediate compound having theformula

This intermediate compound then is reacted with a primary amine havingthe formula R¹NH₂ to provide a compound of structural formula (II).

INTERMEDIATE 1 Preparation of 3-Oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester

Sodium metal (60 mmol, 1.4 g) was suspended in 80 mL (milliliter)toluene under nitrogen. Ethyl acetoacetate (88 mmol (millimole), 11.4 g)was added dropwise, and the resulting mixture was stirred until allsodium was consumed. The reaction mixture then was cooled in an ice bath(0-5° C.) and 2-bromoacetophenone (60 mmol, 12 g) in 140 mL toluene wasadded dropwise. The reaction was stirred overnight with gradual warmingto room temperature. Analysis of the reaction mixture by GC and TLCshowed that the reaction was complete. The reaction mixture then waswashed with 200 mL water, then the organic and aqueous layers wereseparated. The organic layer was dried over MgSO₄ (magnesium sulfate).The toluene was removed from the organic layer under vacuum to provide ayellow oil which was purified by Kugelrohr distillation (b.p. 135-145°C. at 0.35 Torr.).

INTERMEDIATE 2 Preparation of 2-Benzoyl-4-oxo-pentanoic acid ethyl ester

Potassium carbonate (200 mmol, 27.6 g) was added to a solution of ethylbenzoylacetate (10 mmol, 19.2 g) and potassium iodide (20 mmol, 3.3 g)in dry acetone (100 mL). The resulting solution was refluxed, andchloroacetone (110 mmol, 10.7 g) was added dropwise. Analysis of thereaction mixture by GC/MS indicated the presence of Intermediate 2.Solid material was filtered from the reaction mixture, then washed with200 mL acetone. The resulting filtrate was concentrated under vacuum,redissolved in ether, then washed three times with water (100 mL). Theorganic layer was separated from the aqueous layer, and dried overMgSO₄. The solids were separated, and the organic solvents removed undervacuum to yield a yellow/orange oil. The crude oil was purified byKugelrohr distillation (b.p. 175-185 at 0.65 Torr.).

EXAMPLE 1 Preparation of1-(3-Dimethylaminophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

N,N-dimethyl-1,3-phenylenediamine dihydrochloride (5 mmol, 1.09),triethylamine (10 mmol, 1.4 mL), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combinedin ethanol (25 mL), then heated under reflux (12 hrs). The solvents wereremoved under vacuum, the resulting oil was redissolved in methylenechloride, then applied to a silica column. The purified product wascollected as a translucent oil. The named compound was identified by¹H-NMR (CDCl₃, ppm) 1.28 t (3H) , 2.35 s (3H) , 2.77 s (6H) , 4.2 q (2H), 6.3-6.7 m (4H), and 7.0-7.3 m (5H).

EXAMPLE 2 Preparation of1-(3-Chlorophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Chloroaniline (5 mmol, 0.6 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 12 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. Upon completion of the reaction, the crudesolid was recrystallized from ethanol to provide the named product (m.p.93.2° C.-94.6° C.).

EXAMPLE 3 Preparation of1-(3-Chlorophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Chloroaniline (5 mmol, 0.6 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. Upon completion of the reaction,the crude solid was recrystallized from ethanol to afford the desiredproduct (m.p. 99.6° C.-100.5° C.).

EXAMPLE 4 Preparation of5-Methyl-2-phenyl-1-m-tolyl-1H-pyrrole-3-carboxylic acid ethyl ester

m-Toluidine (5 mmol, 0.5 g), 2-benzoyl-4-oxo-pentanoic acid ethyl ester(5 mmol, 1.2 g), and tosic acid (0.1 g) were combined in ethanol, thenheated under reflux. The resulting oil was resuspended in methylenechloride, then washed with water, 5% sodium bicarbonate, and brine. Theorganic layer was dried over NaSO₄ (sodium sulfate), the solids wereseparated, and the solvents evaporated under vacuum. The crude productwas recrystallized from ethanol to yield the named product (m.p. 92.5°C.-94.0° C.).

EXAMPLE 5 Preparation of2-Methyl-5-phenyl-l-m-tolyl-1H-pyrrole-3-carboxylic acid ethyl ester

m-Toluidine (5 mmol, 0.5 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acidethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting oil was resuspended inmethylene chloride, then washed with water, 5% sodium bicarbonate, andbrine. The organic layer was dried over NaSO₄, then the solids wereseparated and the solvents evaporated under vacuum. The crude productwas recrystallized from ethanol to yield the named product (m.p. 103°C.-104.5° C.).

EXAMPLE 6 Preparation of1-(3-Bromophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Bromoaniline (5 mmol, 0.9 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), and tosic acid were combined in ethanol, thenheated under reflux. The resulting oil was resuspended in methylenechloride, then washed with water, 5% sodium bicarbonate and brine. Theorganic layer was dried over MgSO₄, then the solids were separated andthe solvents evaporated under vacuum. The crude product wasrecrystallized from ethanol to yield the named product (m.p. 117.5°C.-119.2° C.).

EXAMPLE 7 Preparation of1-(3-Acetylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

3′-Amino-acetophenone (5 mmol, 0.7 g), 2-benzoyl-4-oxo-pentanoic acidethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The residual oil was purified oversilica and yielded the named product (m.p. 100° C.-102° C.).

EXAMPLE 8 Preparation of1-(3-Acetylphenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

3′-Aminoacetophenone (5 mmol, 0.7 g),3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acid ethyl ester (5 mmol, 1.2 g),and tosic acid (0.1 g) were combined in ethanol, then heated underreflux. The resulting oil was purified over silica and yielded the namedproduct as a solid (m.p. 102° C.-104° C.).

EXAMPLE 9 Preparation of1-(3-Methanesulfonylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

3-Methylsulfonylaniline hydrochloride (5.8 mmol, 1.2 g),2-benzoyl-4-oxo-pentanoic acid ethyl ester (5.8 mmol, 144 g),triethylamine (5.8 mmol, 0.61 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The resulting oil was resuspendedin chloroform, then washed with water, 5% sodium bicarbonate, and brine.The organic layer was dried over NaSO₄ ₄, then the solids were separatedand solvents removed under vacuum. The crude material was purified oversilica to yield the named product (m.p. 131° C.-133° C.).

EXAMPLE 10 Preparation of1-(3-Methanesulfonylphenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

3-Methylsulfonylaniline (1.4 mmol, 0.3 g),3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acid ethyl ester (1.4 mmol, 0.35g), triethylamine (1.4 mmol, 0.14 g), and tosic acid (0.1 g) werecombined in ethanol, then heated under reflux. The resulting oil wasresuspended in chloroform, then washed with water, 5% sodiumbicarbonate, and brine. The organic layer was dried over NaSO₄, then thesolids were separated and the solvents removed under vacuum. The crudematerial was purified over silica gel to yield the named product as anoil. The structure of the compound was verified by ¹H-NMR (CDCl₃, ppm):1.36 t (3H), 2.45 s (3H), 2.82 s (3H), 4.3 q (2H), and 6.8-7.2 m (9H).

EXAMPLE 11 Preparation of1-(3-Carbamoylphenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

3-Aminobenzamide (5 mmol, 1.0 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The resulting precipitate wasfiltered from the mixture, then washed with ethanol. The white solidproduct was used without further purification (m.p. 237.8° C.-238.8°C.).

EXAMPLE 12 Preparation of1-(3-Carbamoylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

3-Aminobenzamide (5 mmol, 1.0 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. The resulting precipitate was filtered fromthe reaction mixture, then washed with ethanol. The tan solid productwas used without further purification (m.p. 208.9° C.-210.3° C.).

EXAMPLE 13 Preparation of1-(3-Dimethylaminophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

N,N-Dimethyl-1,3-phenylenediamine dihydrochloride (5 mmol, 1.0 g),2-benzoyl-4-oxo-pentanoic acid ethyl ester (5 mmol, 1.2 g),triethylamine (10 mmol, 10 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting oil was resuspended inmethylene chloride, then washed with water, 5% sodium bicarbonate, andbrine. The organic layer was dried over NaSO₄, then the solids wereseparated and the solvents removed under vacuum. The crude material waspurified over silica gel and yielded the named product (m.p. 111.7°C.-114° C.).

EXAMPLE 14 Preparation of1-(3-Iodophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Iodoaniline (5 mmol, 1.1 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. The residual oil was resuspended in methylenechloride, then washed with water, 5% sodium bicarbonate, and brine. Thesolids were separated, and the solvents removed under vacuum. Theresulting solid was recrystallized in ethanol. A portion of the materialdid not dissolve, and was separated by filtration. Both therecrystallized and filtered material yielded the named product (m.p.142.6° C.-144° C).

EXAMPLE 15 Preparation of2-Methyl-5-phenyl-l-(3-sulfophenyl)-1H-pyrrole-3-carboxylic acid ethylester

Metanilic acid (5 mmol, 0.9 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), sodium hydroxide (5 mmol, 0.2 g), andtosic acid (0.1 g) were combined in ethanol, then heated under reflux.The unreacted metanilic acid was precipitated on ice and filtered fromthe reaction mixture. The filtrate was evaporated to yield an oil, whichwas purified over silica gel. After standing, the named productsolidified as purple crystals (m.p. 94 C.-96° C.).

EXAMPLE 16 Preparation of5-Methyl-2-phenyl-l-(3-sulfophenyl)-1H-pyrrole-3-carboxylic acid ethylester

Metanilic acid (5 mmol, 0.2 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), sodium hydroxide (5 mmol, 0.2 g), and tosic acid(0.1 g) were combined in ethanol, then heated under reflux. Theresulting oil was treated with HCl (hydrochloric acid) at 0° C. to 5° C.to precipitate the unreacted metanilic acid. The remaining material wasconcentrated under vacuum and purified over silica gel. The namedproduct was collected as brown crystals (m.p. 91° C.-92.5° C.)

EXAMPLE 17 Preparation of1-(3-Fluorophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Fluoroaniline (5 mmol, 0.6 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. The resulting oil was resuspended inchloroform, then washed with water, 5% sodium bicarbonate, and brine.The organic layer was dried over NaSO₄, then the solids filtered awayand the solvents removed under vacuum. The named product wasrecrystallized from ethanol (m.p. 100° C.-101° C.).

EXAMPLE 18 Preparation of1-(3-Fluorophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Fluoroaniline (5 mmol, 0.6 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid were combined inethanol, then heated under reflux. The resulting solid was resuspendedin chloroform, then washed with water, 5% sodium bicarbonate, and brine.The organic layer was dried over NaSO₄, then the solids were separatedand the solvents removed under vacuum. The named product wasrecrystallized from ethanol (m.p. 96.3° C.-98.0° C.).

EXAMPLE 19 Preparation of2-Methyl-5-phenyl-1-pyridin-3-yl-1H-pyrrole-3-carboxylic acid ethylester

3-Aminopyridine (5 mmol, 0.5 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g) and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting oil was resuspended inether, then washed with water and brine. The organic layer was driedover MgSO₄, then the solids were separated and the solvents were removedunder vacuum. The named product was purified over silica gel andanalyzed by ¹H-NMR (CDCl₃, ppm): 1.35 t (3H), 2.40 s (3H), 4.3 q (2H),6.78 s (1H), 7.03-7.39 m (7H), 8.4 sd (1H), and about 8.6 dd (1H).

EXAMPLE 20 Preparation of1-Cyclohexyl-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester

Cyclohexylamine (5 mmol, 0.5 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g) and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting residue was resuspendedin ether, then washed with water and brine. The organic layer was driedover MgSO₄, and the solids were separated and the solvents removed undervacuum. Purification of the oil yielded the named product, ¹H-NMR(CDCl₃₁ ppm): 1.0 bm (2H), 1.23 t (3H), 1.6-1.7 bm (8H), 2.66 s (3H),4.16 q (2H), 6.39 s (1H), and 7.25 bm (5H).

EXAMPLE 21 Preparation of 5-Methyl-1,2-diphenyl-1H-pyrrole-3-carboxylicacid ethyl ester

Aniline (10 mmol, 0.9 g), 2-benzoyl-4-oxo-pentanoic acid ethyl ester (10mmol, 2.5 g), and tosic acid (0.1 g) were combined in ethanol, thenheated under reflux. The named product was recrystallized from ethanol(m.p. 130° C.-131.7° C.).

EXAMPLE 22 Preparation of2-Methyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Nitroaniline (10 mmol, 1.4 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (10 mmol, 2.5 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The named product wasrecrystallized from ethanol (m.p. 143° C.-145° C.).

EXAMPLE 23 Preparation of5-Methyl-1-(5-nitropyridin-2-yl)-2-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

5-Nitro-2-aminopyridine (5 mmol, 0.7 g), 2-benzoyl-4-oxo-pentanoic acidethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting solid was trituratedwith chloroform, and the insoluble material filtered from the reactionmixture. The filtrate was purified over silica gel and yielded the namedproduct (m.p. 146.6° C.-148.6° C.).

EXAMPLE 24 Preparation of1-(3-Carboxyphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

3-Aminobenzoic acid (10 mmol, 1.4 g), 2-benzoyl-4-oxo-pentanoic acidethyl ester (10 mmol, 2.5 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The resulting oil was resuspended inchloroform-ether, then the insoluble material was filtered from thereaction product. The soluble material was purified over silica gelwhich yielded the named product (m.p. 161.0° C.-162.7° C.).

EXAMPLE 25 Preparation of1-(3-Methoxycarbonylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

Methyl 3-aminobenzoate (10 mmol, 1.4 g), 2-benzoyl-4-oxo-pentanoic acidethyl ester (10 mmol, 2.5 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The named product was purified oversilica gel and crystallized upon standing, ¹H-NMR (CDCl₃, ppm): 1.15 t(3H), 2.08 s (3H), 3.88 s (3H), 4.05 q (2H), 6.56 s (1H), 7.15-7.33 bm(7H), 7.79-7.97 bm (2H).

EXAMPLE 26 Preparation of1-(3-Bromophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Bromoaniline (5 mmol, 0.9 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The named product wasrecrystallized from ethanol (m.p. 111° C.-112.7° C.).

EXAMPLE 27 Preparation of1-(3-Iodophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Iodoaniline (5 mmol, 1.1 g), 3-Oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The named product wasrecrystallized in ethanol (m.p. 111° C.-112.9° C.).

EXAMPLE 28 Preparation of2-Methyl-5-phenyl-1-(3-trifluoro-methylphenyl)-1H-pyrrole-3-carboxylicacid ethyl ester

m-Trifluoromethylaniline (5 mmol, 0.8 g),3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acid ethyl ester (5 mmol, 1.2 g),and tosic acid (0.1 g) were combined in ethanol, then heated underreflux. The crude material was resuspended in chloroform and washed withwater, then with brine. The organic layer was dried over NaSO₄. Thesolids were separated and the solvents removed under vacuum. Theresulting oil was purified over silica gel to yield the named product,¹H-NMR (CDCl₃, ppm): 1.37 t (3H), 2.42 s (3H), 4.32 q (2H), 6.80 s (1H),7.0-7.6 bm (9H).

EXAMPLE 29 Preparation of5-Methyl-2-phenyl-1-(3-trifluoro-methylphenyl)-1H-pyrrole-3-carboxylicacid ethyl ester

m-Trifluoromethylaniline (5 mmol, 0.8 g), 2-benzoyl-4-oxo-pentanoic acidethyl ester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined inethanol, then heated under reflux. The crude material was resuspended inchloroform, washed with water, then washed with brine. The organic layerwas dried over NaSO₄. The solids were separated and the solvents removedunder vacuum. The resulting oil was purified over silica gel to yieldthe named product, ¹H-NMR (CDCl₃, ppm): 1.01 t (3H), 1.98 b (3H), 4.00 q(2H), 6.48 s (1H), 7.00-7.33 bm (9H).

EXAMPLE 30 Preparation of2-Methyl-1-(3-nitrobenzyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Nitrobenzylamine hydrochloride (3 g) was suspended in an aqueoussolution of saturated sodium bicarbonate (30 mL), then extracted withchloroform. The organic layer was dried over MgSO₄, the solids separatedand solvents removed under vacuum to yield the free base. Them-nitrobenzylamine (5 mmol, 0.8 g) prepared above,3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acid ethyl ester (5 mmol, 1.2 g),and tosic acid (0.1 g) were combined in ethanol, then heated underreflux. The residue was resuspended in ethyl ether, and the benzylaminetosic acid salt was filtered from the mixture. The filtrate wasconcentrated under vacuum, and the resulting oil purified over silica toyield the named product, ¹H-NMR (CDCl₃, ppm): 1.24 t (3H), 2.36 s (3H),4.07-4.30 q (2H), 5.11 s (2H), 6.58 s (1H), 6.98-7.44 bm (7H), 7.69 bs(1H) , 7.97 bd (1H).

EXAMPLE 31 Preparation of5-Methyl-1-(3-nitrobenzyl)-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

m-Nitrobenzylamine hydrochloride (3 g) was suspended in an aqueoussolution of saturated sodium bicarbonate (30 mL), and extracted withchloroform. The organic layer was dried over MgSO₄, the solids saturatedand solvents removed under vacuum to yield the free base. Them-nitrobenzylamine (5 mmol, 0,8 g) prepared above,2-benzoyl-4-oxo-pentanoic acid ethyl ester (5 mmol, 1.2 g), and tosicacid were combined in ethanol, then heated under reflux. The resultingresidue was purified over silica gel to yield the named product, ¹H-NMR(CDCl₃, ppm) 1.10 t (3H), 3.16 s (3H), 4.08 q (2H), 5.04 s (2H), 6.55 s(1H), 7.07-7.53 bm (7H), 7.72 bs (1H), 8.04 bd (1H).

EXAMPLE 32 Preparation of5-Methyl-2-phenyl-1-pyridin-3-yl-1H-pyrrole-3-carboxylic acid ethylester

3-Aminopyridine (5 mmol, 0.5 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (5 mmol, 1.2 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. The resulting oil was purified over silica,but still contained contaminants. This material was triturated inhexane, and the solids were separated by filtration. The solids werewashed with benzene two times to yield the named product (m.p. 102°C.-103° C.).

EXAMPLE 33 Preparation of 2-Methyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester

p-Nitroaniline (5 mmol, 0.7 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (5 mmol, 1.25 g), and tosic acid were combined inethanol, then heated under reflux. The reaction mixture was cooled onice (0-5° C.), and the precipitate filtered from the reaction mixture.¹H-NMR analysis confirmed that the precipitate was the named product,(CDCl₃, ppm); 1.38 t (3H), 2.45 s (3H), 4.20 q (2H), 6.80 s (1H),6.97-7.35 bm (7H), 8.25 d (2H).

EXAMPLE 34 Preparation of2-Methyl-1-(2-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

o-Nitroaniline (15 mmol, 2.1 g), 3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyricacid ethyl ester (15 mmol, 3.7 g), and tosic acid (0.1 g) were combinedin ethanol, then heated under reflux. The resulting oil was purifiedover three silica gel columns to yield the named product, ¹H-NMR (CDCl₃,ppm): 1.36 t (3H), 2.36 s (3H), 4.31 q (2H), 6.80 s (1H), 7.03-7.66 bm(6H), 7.68 dt (2H) 7.97 dd (1H)

EXAMPLE 35 Preparation of2-Methyl-1-(5-nitropyridin-2-yl)-5-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

5-Nitro-pyridin-2-ylamine (5 mmol, 0.7 g),3-oxo-2-(2-oxo-2-phenyl-ethyl)-butyric acid ethyl ester (5 mmol, 1.2 g),and tosic acid (0.1 g) were combined in ethanol, then heated underreflux. The resulting solid was triturated with chloroform, the filtratewas further purified over silica gel to yield the named product, ¹H-NMR(CDCl₃, ppm): 1.35 t (3H), 2.54 s (3H), 4.30 q (2H), 6.78 s (1H),6.96-7.25 bm (6H), 8.33 dd (1H) , 9.39 sd (1H).

EXAMPLE 36 Preparation of5-Methyl-1-(4-nitrophenyl)-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

p-Nitroaniline (10 mmol, 1.4 g), 2-benzoyl-4-oxo-pentanoic acid ethylester (10 mmol, 2.5 g), and tosic acid (0.1 g) were combined in ethanol,then heated under reflux. The reaction mixture was filtered and thesolids washed with ethanol. The collected material was determined to bethe named product (m.p. 190° C.-192° C.).

EXAMPLE 37 Preparation of1-(4-Aminophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

5-Methyl-1-(4-nitrophenyl)-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester of Example 36 (4 mmol, 1.5 g) was dissolved in ethanol, andtreated with 5% Pd/C (0.15 g). The mixture was hydrogenated with H₂(g)at 45 psi. Upon completion of the reaction, the mixture was filteredthrough Celite, and the solvents removed under vacuum. Analysis of theresulting solid showed it was the named product (m.p. 181° C.-183° C.)

EXAMPLE 38 Preparation of1-(4-Aminophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

2-Methyl-1-(4-nitro-phenyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester of Example 33 (2 mmol, 0.8 g) was dissolved 50% solution of ethylacetate in ethanol, and treated with Pd/C (0.088 g). The mixture washydrogenated with H₂(g) at 45 psi. Upon completion of the reaction, themixture was filtered through Celite and the solvents removed undervacuum. This material was purified over silica gel to yield the namedproduct.

EXAMPLE 39 1-(3-Nitrophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

The named product was purchased from Maybridge Chemical Co., Ltd.,Cornwall, UK, and used without further purification.

EXAMPLE 40 Preparation of1-(3-Aminophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester

The compound of Example 39 (0.86 mmol, 0.3 g) was dissolved in methanol,and treated with nickel chloride (NiCl₂) and sodium borohydride (NaBH₄)(17.2 mmol). The methanol was removed, and then the reaction mixture wasresuspended in water, and next extracted with ethyl acetate. Thereaction mixture was dried over MgSO₄, filtered, and concentrated. Thenamed product was purified over silica. ¹H-NMR (CDCl₃, ppm): 1.37 t(3H), 2.41 s (3H), 3.73 s (2H), 4.31 q (2H), 6.43 s (1H), 6.54 d (1H),6.67 dd (1H), 6.78 s (1H), 7.11-7.18 m (5H).

EXAMPLE 41 Preparation of1-(3-Aminophenyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester

The compound of Example 22 (0.86 mmol, 0.3 g) was dissolved in methanoland treated with NiCl₂ and NaBH₄ (17.2 mmol) using the procedure setforth in Example 40. The named product was purified over silica (m.p.142° C.-143° C.).

EXAMPLE 421-(2,3-Dichlorophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acidethyl ester

The named product was purchased from Maybridge Chemical Co., Ltd., andused without further purification.

EXAMPLE 43 Preparation of5-Methyl-1-(3-nitrophenyl)-2-phenyl-1H-pyrrole-3-carboxylic acid

The compound of Example 39 (0.29 mmol, 0.1 g) was dissolved in a 50%dioxane and water, then treated with lithium hydroxide (LiOH, 0.87 mmol,0.0363 g) and heated to at least 50° C. The solvents were removed andthe base extracted with ethyl acetate. The aqueous layer was acidifiedto pH<2 and extracted with ethyl acetate. The organic layer was driedover MgSO₄, the solids filtered, and the solvents removed under vacuum.The named product was used without further purification. ¹H-NMR (CDCl₃,ppm): 2.48 s (3H), 6.88 s (1H), 7.03 dd (2H), 7.18 m (3H), 7.47 dd (1H),7.6 t (1H), 8.08 st (1H), 8.27 dd (1H).

EXAMPLE 44 Preparation of 5-Methyl-1,2-diphenyl-1H-pyrrole-3-carboxylicacid

The compound of Example 21 (0.33 mmol, 0.1 g) was hydrolyzed in the samemanner as set forth in Example 43 to yield the named product, ¹H-NMR(CDCl₃ ppm): 2.44 s (3H), 6.87 s (1H), 7.06 m (2H), 7.15 m (5H), 7.40 m(3H).

EXAMPLE 451-(3,5-Bis-trifluoromethylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

The named product was purchased from Maybridge Chemical Co., Ltd., andused without further purification.

EXAMPLE 46 Preparation of1-(3-Ethoxycarbonylphenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester

This compound was formed in an identical manner as the compound ofExample 25, except ethyl 3-aminobenzoate was used as a startingmaterial. The named product had an m.p. of 108° C.-110° C.

The compounds of structural formulae (I) and (II) were tested for anability to inhibit PDE4. The ability of a compound to inhibit PDE4activity is related to the IC₅₀ value for the compound, i.e., theconcentration of inhibitor required for 50% inhibition of enzymeactivity. The IC₅₀ value for compounds of structural formulae (I) and(II) were determined using human recombinant PDE4.

The compounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE4 of less than about 100 μM, and preferablyless than about 50 μM, and more preferably less than about 25 μm. Thecompounds of the present invention typically exhibit an IC₅₀ valueagainst recombinant human PDE4 of less than about 10 μM, and often lessthan about 1 μM. To achieve the full advantage of the present invention,a present PDE4 inhibitor has an IC₅₀ of about 0.005 μM to about 25 μM.

The IC₅₀ values for the compounds were determined fromconcentration-response curves typically using concentrations rangingfrom 0.1 pM to 500 μM. Tests against other PDE enzymes using standardmethodology, as described in Loughney et al., J. Biol. Chem., 271, pp.796-806 (1996), also showed that compounds of the present invention arehighly selective for the cAMP-specific PDE4 enzyme.

In particular, a compound of the present invention, i.e., Example 22,has an IC₅₀ vs. human recombinant PDE4B of 0.15 μM, but has an IC₅₀ vs.PDE1A of 35.5 μM, vs. PDE1B of 21.0 μM, vs. PDE1C of 21.8 μM, vs. PDE2of 4.5 μM, vs. PDE3A of 89 μM, vs. PDE5 of 12.5 μM, and vs. PDE7 of 10.6μM. This illustrates the selectivity of the present compound withrespect to inhibiting PDE4.

The compounds of structural formulae (I) and (II) also were tested foran ability to reduce TNFα secretion in human peripheral bloodlymphocytes. The ability to reduce TNFα secretion is related to the EC₅₀values (i.e., the effective concentration of the compound capable ofinhibiting 50% of the total TNFα).

The compounds of the present invention typically exhibit an EC₅₀ valueof less than about 100 μM, and preferably less than about 50 μM, andmore preferably less than about 15 μM. The compounds of the presentinvention preferably exhibit a PBL/TNFα EC₅₀ value of less than about 10μM, and often less than about 7 μM. To achieve the full advantage of thepresent invention, a present PDE4 inhibitor has an EC₅₀ value of about 1μM to about 20 μM.

The production of recombinant human PDEs and the IC₅₀ and EC₅₀determinations can be accomplished by well-known methods in the art.Exemplary methods are described as follows:

Expressions of Human PDEs

Expression in Baculovirus-Infected Spodoptera fugiperda (Sf9 g) Cells

Baculovirus transfer plasmids were constructed using either pBlueBacIII(Invitrogen) or pFastBac (BRL-Gibco). The structure of all plasmids wasverified by sequencing across the vector junctions and by fullysequencing all regions generated by PCR. Plasmid pBB-PDE1A3/6 containedthe complete open reading frame of PDE1A3 (Loughney et al., J. Biol.Chem., 271, pp. 796-806 (1996)) in pBlueBacIII. Plasmid Hcam3aBBcontained the complete open reading frame of PDE1C3 (Loughney et al.(1996)) in pBlueBacIII. Plasmid pBB-PDE3A contained the complete openreading frame of PDE3A (Meacci et al., Proc. Natl. Acad. Sci., USA, 89,pp. 3721-3725 (1992)) in pBlueBacIII.

Recombinant virus stocks were produced using either the MaxBac system(Invitrogen) or the FastBac system (Gibco-BRL) according to themanufacturer's protocols. In both cases, expression of recombinant humanPDEs in the resultant viruses was driven off the viral polyhedronpromoter. When using the MaxBac® system, virus was plaque purified twicein order to insure that no wild type (occ+) virus contaminated thepreparation. Protein expression was carried out as follows. Sf9 cellswere grown at 27° C. in Grace's Insect culture medium (Gibco-BRL)supplemented with 10% fetal bovine serum, 0.33% TC yeastolate, 0.33%lactalbumin hydrolysate, 4.2 mM NaHCO₃, 10 μg/mL gentamycin, 100units/mL penicillin, and 100 μg/mL streptomycin. Exponentially growingcells were infected at a multiplicity of approximately 2 to 3 virusparticles per cell and incubated for 48 hours. Cells were collected bycentrifugation, washed with nonsupplemented Grace's medium, andquick-frozen for storage.

Expression in Saccharomyces cerevisiae (Yeast)

Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C, PDE4D,PDE5, and PDE7 was carried out similarly to that described in Example 7of U.S. Pat. No. 5,702,936, incorporated herein by reference, exceptthat the yeast transformation vector employed, which is derived from thebasic ADH2 plasmid described in Price et al., Methods in Enzymology,185, pp. 308-318 (1990), incorporated yeast ADH2 promoter and terminatorsequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2×SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEPmedium-containing glycerol was added to a final concentration of2×YET/3% glycerol. Approximately 24 hr later, cells were harvested,washed, and stored at −70° C.

Calmodulin Purification

Calmodulin used for activation of the PDE1 enzymes was purified frombovine testes essentially as described by Dedman et al., Methods inEnzymology, 102, pp. 1-8 (1983) using the Pharmacia Phenyl-Sepharose®procedure.

Immobilization of Calmodulin on Agarose

Calmodulin was immobilized on BioRad Affi-Gel® 15 per manufacturer'sinstructions.

Human Phosphodiesterase Preparations

Phosphodiesterase Activity Determinations

Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). Alternatively, in assays assessingPDE1-specific activity, incubation mixtures further incorporated the useof 0.1 mM CaCl₂ and 10 μg/mL calmodulin. PDE enzyme was present inquantities that yield <30% total hydrolysis of substrate (linear assayconditions). The assay was initiated by addition of substrate (1 mM[32P]cAMP or cGMP), and the mixture was incubated for 12 minutes.Seventy-five (75) μg of Crotalus atrox venom then was added, and theincubation was continued for 3 minutes (15 minutes total). The reactionwas stopped by addition of 200 μL of activated charcoal (25 mg/mLsuspension in 0.1 M NaH₂PO₄, pH 4). After centrifugation (750×g for 3minutes) to sediment the charcoal, a sample of the supernatant was takenfor radioactivity determination in a scintillation counter and the PDEactivity was calculated.

Inhibitor analyses were performed similarly to the method described inLoughney et al., J. Biol. Chem., 271, pp. 796-806 (1996), except bothcGMP and cAMP were used, and substrate concentrations were kept below 32nM, which is far below the Km of the tested PDE5.

Purification of PDE1A3 from SF9 Cells

Cell pellets (5 g) were mixed with 10 mL of Lysis Buffer (50 mM MOPS pH7.5, 2 mM dithiothreitol (DTT), 2 mM benzamidine HCl, 5 μM ZnSO₄, 0.1 mMCaCl₂, 20 μg/mL calpain inhibitors I and II, and 5 μg/mL each ofleupeptin, pepstatin, and aprotinin) at room temperature. The cells werelysed by passage through a French® pressure cell (SLM-Aminco®,Spectronic Instruments, Inc., Rochester N.Y.). The resultant lysate wascentrifuged in a Beckman ultracentrifuge using a type T180 rotor at45,000 rpm for 1 hr. The supernatant was recovered and filtered througha 0.2 m filter. This filtrate was applied to a 2.6×90 cm column ofSEPHACRYL® S-300 equilibrated in Column Buffer A (Lysis Buffercontaining 100 mM NaCl, and 2 mM MgCl₂). The column flow rate wasadjusted to 1 mL/min and fractions of 7 mL were collected. Activefractions were pooled and supplemented with 0.16 mg of calmodulin. Theenzyme was applied overnight at a flow rate of 0.2 mL/min to an ACC.-1agarose immunoaffinity column as described in Hansen et al., Methods inEnzymology 159, pp. 453-557 (1988). The column was washed with 5 volumesof Column Buffer B (Column Buffer A without NaCl) and followed by 5volumes of Column Buffer C (Column Buffer A containing 250 mM NaCl). Thecolumn was eluted with Column Buffer D (50 mM MOPS pH 7.5, 1 mM EDTA, 1mM EGTA, 1 mM DTT, 1 mM benzamidine HCl, 100 mM NaCl, 20 μg/mL calpaininhibitors I and II, and 5 μg/mL each of leupeptin, pepstatin, andaprotinin) by applying one column volume at 0.1 mL/min, stopping flowfor 1 hour, and then continuing elution at the same flow rate. Fractionsof 0.5 mL were collected. Fractions displaying activity were pooled, andfirst dialyzed against dialysis buffer containing 25 mM MOPS pH 7.5, 100mM NaCl, 10 μM ZnSO₄, 1 mM CaCl₂, 1 mM DTT, and 1 mM benzamidine HCl. Asubsequent dialysis against dialysis buffer containing 50% glycerol wasperformed prior to quick-freezing the sample with dry ice and storage at−70° C. The resultant preparations were about 10 to 15% pure bySDS-PAGE. These preparations had specific activities of about 5 to 20μmol cAMP hydrolyzed per minute per milligram protein.

Purification of PDE1B from S. cerevisiae

Yeast cells (50 g) were thawed by mixing with 100 mL glass beads (0.5mM, acid washed) and 200 mL Buffer A at room temperature. Buffer Aconsisted of 50 mM MOPS pH 7.5, 1 mM DTT, 2 mM benzamidine HCl, 0.01 mMZnSO₄, 5 mM MgCl₂, 20 μg/mL calpain inhibitors I and II, and 5 μg/mLeach of leupeptin, pepstatin, and aprotinin. The mixture was cooled to4° C., transferred to a Bead-Beater®, and the cells lysed by rapidmixing for 6 cycles of 30 seconds each. The homogenate was centrifugedfor 15 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at9,000 rpm and 4° C. The supernatant was recovered and centrifuged in aBeckman XL-80 ultracentrifuge using a TI45 rotor at 36,000 rpm for 45minutes at 4° C. The supernatant was recovered and PDE1B wasprecipitated by the addition of solid ammonium sulfate (0.33 g/mLsupernatant) while stirring in an ice bath and maintaining the pHbetween 7.0 and 7.5. This mixture then was centrifuged for 22 minutes ina Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000×g). Thesupernatant was discarded and the pellet was dissolved in 100 mL ofbuffer B (50 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mMZnSO₄, 2 mM MgCl₂, 2 mM CaCl₂, and 5 μg/mL each of leupeptin, pepstatin,and aprotinin). The pH and conductivity were corrected to 7.5 and 15-20milliSiemens (mS), respectively. This solution was loaded onto a 20 mLcolumn of calmodulin-Agarose that had been equilibrated with 10 columnvolumes of Buffer B at a rate of 1 mL/min. The flow-through wasreapplied to the column at least 5 times. The column was washed with 5volumes of Buffer B, 5 volumes of buffer B containing 250 mM NaCl, and 2volumes of Buffer B without NaCl again. Elution was accomplished byapplying one volume of Buffer C (50 mM MOPS pH 7.5, 1 mM EDTA, 1 mMEGTA, 1 mM DTT, 1 mM benzamidine HCl) at 0.33 mL/min, then stopping flowfor 1 hour before continuing the elution. Fractions of about 4 mL werecollected and assayed for PDE activity. Active fractions were pooled andconcentrated to a volume of 5 mL, using an Amicon ultrafiltrationsystem. The concentrate was then applied to a 320 mL Sephacryl® S-300column (1.6×150 cm) that had been equilibrated with at least 2 volumesof Buffer D (25 mM MOPS pH 7.5, 1 mM DTT, 1 mM benzamidine HCl, 0.01 mMZnSO₄, 2 mM CaCl₂, and 100 mM NaCl). The column was developed at a flowrate of 1 mL/min (11 cm/hr), and 5 mL fractions were collected. Theactivity peak was pooled and dialyzed overnight against Buffer Dcontaining 50% glycerol. The purified enzyme was frozen on dry ice andstored at −70° C. The resultant preparations were about >90% pure bySDS-PAGE. These preparations had specific activities of about 10 to 30μmol cGMP hydrolyzed per minute per milligram protein.

Purification of PDE1C3 from Sf9 Cells

Cell pellets (5 g) were thawed on ice with 20 mL of Lysis Buffer (50 mMMOPS pH 7.4, 10 μM ZnSO₄, 0.1 mM CaCl₂, 1 mM DTT, 2 mM benzamidine HCl,5 μg/mL each of pepstatin, leupeptin, and aprotinin). Cells were lysedby passage through a French® pressure cell (SLM-Aminco®, SpectronicInstruments) while temperatures were maintained below 10° C. Theresultant cell homogenate was centrifuged at 36,000 rpm at 4° C. for 45min in a Beckman ultracentrifuge using a Type TI45 rotor. Thesupernatant was discarded and the resultant pellet was resuspended with40 mL of Solubilization Buffer (Lysis Buffer containing 1 M NaCl, 0.1 MMgCl₂, 1 mM CaCl₂, 20 μμg/mL calmodulin, and 1% Sulfobetaine SB12(Z3-12) by sonicating using a VibraCell tuner with a microtip for 3×30seconds. This was performed in a crushed ice/salt mix for cooling.Following sonication, the mixture was slowly mixed for 30 minutes at 4°C. to finish solubilizing membrane bound proteins. This mixture wascentrifuged in a Beckman ultracentrifuge using a type TI45 rotor at36,000 rpm for 45 minutes. The supernatant was diluted with Lysis Buffercontaining 10 μg/mL calpain inhibitors I and II. The precipitatedprotein was centrifuged for 20 minutes at 9,000 rpm in a Beckman JA-10rotor. The recovered supernatant then was subjected to Mimetic Blue® APAgarose Chromatography.

To run the Mimetic Blue® AP Agarose Column, the resin initially wasshielded by the application of 10 bed volumes of 1% polyvinylpyrrolidone(i.e., MW of 40,000) to block nonspecific binding sites. The looselybound PVP-40 was removed by washing with 10 bed volumes of 2 M NaCl, and10 mM sodium citrate pH 3.4. Just prior to addition of the solubilizedPCE1C3 sample, the column was equilibrated with 5 bed volumes of ColumnBuffer A (50 mM MOPS pH 7.4, 10 μM ZnSO₄, 5 mM MgCl₂, 0.1 mM CaCl₂, 1 mMDTT, 2 mM benzamidine HCl).

The solubilized sample was applied to the column at a flow rate of 2mL/min with recycling such that the total sample was applied 4 to 5times in 12 hours. After loading was completed, the column was washedwith 10 column volumes of Column Buffer A, followed by 5 column volumesof Column Buffer B (Column Buffer A containing 20 mM 5′-AMP), andfollowed by 5 column volumes of Column Buffer C (50 mM MOPS pH 7.4, 10μM ZnSO₄, 0.1 mM CaCl₂, 1 mM DTT, and 2 mM benzamidine HCl). The enzymewas eluted into three successive pools. The first pool consisted ofenzyme from a 5-bed volume wash with Column Buffer C containing 1 mMcAMP. The second pool consisted of enzyme from a 10-bed volume wash withColumn Buffer C containing 1 M NaCl. The final pool of enzyme consistedof a 5-bed volume wash with Column Buffer C containing 1 M NaCl and 20mM cAMP.

The active pools of enzyme were collected and the cyclic nucleotideremoved via conventional gel filtration chromatography or chromatographyon hydroxyapatite resins. Following removal of cyclic nucleotides, theenzyme pools were dialyzed against Dialysis Buffer containing 25 mM MOPSpH 7.4, 10 μM ZnSO₄, 500 mM NaCl, 1 mM CaCl₂, 1 mM DTT, 1 mM benzamidineHCl, followed by dialysis against Dialysis buffer containing 50%glycerol. The enzyme was quick-frozen with the aid of dry ice and storedat −70° C.

The resultant preparations were about >90% pure by SDS-PAGE. Thesepreparations had specific activities of about 0.1 to 1.0 μmol cAMPhydrolyzed per minute per milligram protein.

Purification of PDE2 from S. cerevisiae

Frozen yeast cell pellets from strain YI34 (10 g, stored at −70° C.)were allowed to thaw on ice in 25 mL of Lysis Buffer (50 mM MOPS, pH7.2, 1 mM EDTA, 1 mM EGTA, 0.1 mM DTT, 0.1 mM4-(2-amino-ethyl)benzenesulfonyl fluoride (AEBSF), 1 μg/mL of pepstatin,leupeptin, aprotinin, calpain inhibitors I and II, and 2 mMbenzamidine). Cells were lysed by three passages through a French®pressure cell (SLM-Aminco®, Spectronic Instruments). The lysate wascentrifuged at 36,000 rpm in a Beckman Ultracentrifuge rotor Type 45Tifor 60 minutes at 4° C. The supernatant was separated from sediment andpassed through a 15 mL Epoxy-cGMP Sepharos® resin at 4° C. two times atabout 0.5 mL/min. The column subsequently was washed with 45 mL of WashBuffer 1 (50 mM MOPS, pH 7.2, 0.1 mM EDTA, 0.1 mM DTT). Following thiswash, the column was washed with 45 mL of Wash Buffer 2 (Wash Buffer 1containing 0.5 M NaCl). Following this salt wash, the column was washedwith 15 mL of Wash Buffer 3 (Wash Buffer 1 containing 0.25 M NaCl). Thecolumn was transferred to room temperature and allowed to warm.Approximately 25 mL of Elution Buffer (Wash Buffer 3 containing 10 mMcGMP, maintained at room temperature) was applied to the column and theeffluent was collected in 2 mL fractions. Small aliquots of each of thefractions were diluted 20-fold in PBS containing 5 mM MgCl₂ to allowhydrolysis of the competing ligand and to aid detection of PDE2activity. Active fractions were passed through a Pharmacia PD-10® gelfiltration column to exchange into Wash Buffer 3. This exchanged poolwas diluted 50% v/v with sterile 80% glycerol and stored at −20° C. Theresultant preparations were greater than 85% pure as judged by SDS-PAGEwith subsequent staining of protein by Coomassie R-250. Thesepreparations had specific activities of about 150 to 250 μmol cGMPhydrolyzed per minute per milligram protein.

Preparation of PDE3A from Sf9 Cells

Cells (2×1010) were suspended in Lysis Buffer containing 50 mM MOPS pH7.5, 2 mM DTT, 2 mM benzamidine HCl, 5 μM ZnSO₄, 0.1 mM CaCl₂, 20 μg/mLcalpain inhibitors I and II, and 5 μg/mL each of leupeptin, pepstatin,and aprotinin. The mixture was sonicated twice for 30 seconds and thecells were lysed in a French® pressure cell (SLM-Aminco®, SpectronicInstruments) at 4° C. The lysate was centrifuged 100,000×g for 45minutes. The pellet was washed once in Lysis Buffer and suspended in 46mL Lysis Buffer with a Dounce homogenizer. Aliquots were stored at −70°C. These preparations had specific activities of about 1 to 2 nmol cAMPhydrolyzed per minute per milligram protein.

Human PDE4A, 4B, 4C, 4D Preparations

Preparation of PDE4A from S. cerevisiae

Yeast cells (50 g of yeast strain YI26 harboring HDUN1.46) were thawedat room temperature by mixing with 50 mL of Lysis Buffer (50 mM MOPS pH7.5, 10 μM ZnSO₄, 2 mM MgCl₂, 14.2 mM 2-mercaptoethanol, 5 μg/mL each ofpepstatin, leupeptin, aprotinin, 20 μg/mL each of calpain inhibitors Iand II, and 2 mM benzamidine HCl). Cells were lysed in a French®pressure cell (SLM-Aminco®, Spectronic Instruments) at 10° C. Theextract was centrifuged in a Beckman JA-10 rotor at 9,000 rpm for 22minutes at 4° C. The supernatant was removed and centrifuged in aBeckman TI45 rotor at 36,000 rpm for 45 minutes at 4° C.

PDE4A was precipitated from the high-speed supernatant by the additionof solid ammonium sulfate (0.26 g/mL supernatant) while stirring in anice bath and maintaining the pH between 7.0 and 7.5. The precipitatedproteins containing PDE4A were collected via centrifugation in a BeckmanJA-10 rotor at 9,000 rpm for 22 minutes. The precipitate was resuspendedin 50 mL of Buffer G (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5 mM MgCl₂, 100 mMNaCl, 14.2 mM 2-mercaptoethanol, 2 mM benzamidine HCl, 5 μg/mL each ofleupeptin, pepstatin, and aprotinin, and 20 μg/mL each of calpaininhibitors I and II) and passed through a 0.45 μm filter.

The resuspended sample (50 to 100 mL) was loaded onto a 5×100 cm columnof Pharmacia SEPHACRYL® S-300 equilibrated in Buffer G. Enzyme activitywas eluted at a flow rate of 2 mL/min and pooled for laterfractionation.

The PDE4A isolated from gel filtration chromatography was applied to a1.6×20 cm column of Sigma Cibacron Blue Agarose-type 300 (10 mL)equilibrated in Buffer A (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5 mM MgCl₂,14.2 mM 2-mercaptoethanol, and 100 mM benzamidine HCl). The column waswashed in succession with 50 to 100 mL of Buffer A, 20 to 30 mL ofBuffer A containing 20 mM 5′-AMP, 50 to 100 mL of Buffer A containing1.5 M NaCl, and 10 to 20 mL of Buffer C (50 mM Tris HCl pH 8, 10 μMZnSO₄, 14.2 mM 2-mercaptoethanol, and 2 mM benzamidine HCl). The enzymewas eluted with 20 to 30 mL of Buffer C containing 20 mM cAMP.

The PDE activity peak was pooled, and precipitated with ammonium sulfate(0.33 g/mL enzyme pool) to remove excess cyclic nucleotide. Theprecipitated proteins were resuspended in Buffer X (25 mM MOPS pH 7.5, 5μM ZnSO₄, 50 mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl), and desaltedvia gel filtration on a Pharmacia PD-10® column per manufacturer'sinstructions. The enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

The resultant preparations were about >80% pure by SDS-PAGE. Thesepreparations had specific activities of about 10 to 40 μmol cAMPhydrolyzed per minute per milligram protein.

Preparation of PDE4B from S. cerevisiae

Yeast cells (150 g of yeast strain YI23 harboring HDUN2.32) were thawedby mixing with 100 mL glass beads (0.5 mM, acid washed) and 150 mL LysisBuffer (50 mM MOPS pH 7.2, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mMbenzamidine HCl, 5 μg/mL each of pepstatin, leupeptin, aprotinin,calpain inhibitors I and II) at room temperature. The mixture was cooledto 4° C., transferred to a Bead-Beater®, and the cells lysed by rapidmixing for 6 cycles of 30 seconds each. The homogenate was centrifugedfor 22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at9,000 rpm and 4° C. The supernatant was recovered and centrifuged in aBeckman XL-80 ultra-centrifuge using a TI45 rotor at 36,000 rpm for 45minutes at 4° C. The supernatant was recovered and PDE4B wasprecipitated by the addition of solid ammonium sulfate (0.26 g/mLsupernatant) while stirring in an ice bath and maintaining the pHbetween 7.0 and 7.5. This mixture was then centrifuged for 22 minutes ina Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000×g). Thesupernatant was discarded and the pellet was dissolved in 200 mL ofBuffer A (50 mM MOPS pH 7.5, 5 mM MgCl₂, 1 mM DTT, 1 mM benzamidine HCl,and 5 μg/mL each of leupeptin, pepstatin, and aprotinin). The pH andconductivity were corrected to 7.5 and 15-20 mS, respectively.

The resuspended sample was loaded onto a 1.6×200 cm column (25 mL) ofSigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A. Thesample was cycled through the column 4 to 6 times over the course of 12hours. The column was washed in succession with 125 to 250 mL of BufferA, 125 to 250 mL of Buffer A containing 1.5 M NaCl, and 25 to 50 mL ofBuffer A. The enzyme was eluted with 50 to 75 mL of Buffer E (50 mM TrisHCl pH 8, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and 20mM cAMP) and 50 to 75 mL of Buffer E containing 1 M NaCl. The PDEactivity peak was pooled, and precipitated with ammonium sulfate (0.4g/mL enzyme pool) to remove excess cyclic nucleotide. The precipitatedproteins were resuspended in Buffer X (25 mM MOPS pH 7.5, 5 μM ZnSO₄, 50mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desalted via gelfiltration on a Pharmacia PD-10® column per manufacturer's instructions.The enzyme pool was dialyzed overnight against Buffer X containing 50%glycerol. This enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

The resultant preparations were about >90% pure by SDS-PAGE. Thesepreparations had specific activities of about 10 to 50 μmol cAMPhydrolyzed per minute per milligram protein.

Preparation of PDE4C from S. cerevisiae

Yeast cells (150 g of yeast strain YI30 harboring HDUN3.48) were thawedby mixing with 100 mL glass beads (0.5 mM, acid washed) and 150 mL LysisBuffer (50 mM MOPS pH 7.2, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mMbenzamidine HC1, 5 μg/mL each of pepstatin, leupeptin, aprotinin,calpain inhibitors I and II) at room temperature. The mixture was cooledto 4° C., transferred to a BEAD-BEATER®, and the cells lysed by rapidmixing for 6 cycles of 30 sec each. The homogenate was centrifuged for22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000rpm and 4° C. The supernatant was recovered and centrifuged in a BeckmanXL-80 ultra-centrifuge using a TI45 rotor at 36,000 rpm for 45 minutesat 4° C.

The supernatant was recovered and PDE4C was precipitated by the additionof solid ammonium sulfate (0.26 g/mL supernatant) while stirring in anice bath and maintaining the pH between 7.0 and 7.5. Thirty minuteslater, this mixture was centrifuged for 22 minutes in a Beckman J2centrifuge using a JA-10 rotor at 9,000 rpm (12,000×g). The supernatantwas discarded and the pellet was dissolved in 200 mL of Buffer A (50 mMMOPS pH 7.5, 5 mM MgCl₂, 1 mM DTT, 2 mM benzamidine HCl, and 5 μg/mLeach of leupeptin, pepstatin, and aprotinin). The pH and conductivitywere corrected to 7.5 and 15-20 mS, respectively.

The resuspended sample was loaded onto a 1.6×20 cm column (25 mL) ofSigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A. Thesample was cycled through the column 4 to 6 times over the course of 12hours. The column was washed in succession with 125 to 250 mL of BufferA, 125 to 250 mL of Buffer A containing 1.5 M NaCl, and then 25 to 50 mLof Buffer A. The enzyme was eluted with 50 to 75 mL of Buffer E (50 mMTris HCl pH 8, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and20 mM cAMP) and 50 to 75 mL of Buffer E containing 1 M NaCl. The PDE4Cactivity peak was pooled, and precipitated with ammonium sulfate (0.4g/mL enzyme pool) to remove excess cyclic nucleotide. The precipitatedproteins were resuspended in Buffer X (25 mM MOPS pH 7.2, 5 μM ZnSO₄, 50mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desalted via gelfiltration on a Pharmacia PD-10® column per manufacturer's instructions.The enzyme pool was dialyzed overnight against Buffer X containing 50%glycerol. This enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

The resultant preparations were about >80% pure by SDS-PAGE. Thesepreparations had specific activities of about 10 to 20 μmol cAMPhydrolyzed per minute per milligram protein.

Preparation of PDE4D from S. cerevisiae

Yeast cells (100 g of yeast strain YI29 harboring HDUN4.11) were thawedby mixing with 150 mL glass beads (0.5 mM, acid washed) and 150 mL LysisBuffer (50 mM MOPS pH 7.2, 10 μM ZnSO₄, 2 mM MgCl₂, 14.2 mM2-mercaptoethanol, 2 mM benzamidine HCl, 5 μg/mL each of pepstatin,leupeptin, aprotinin, calpain inhibitors I and II) at room temperature.The mixture was cooled to 4° C., transferred to a Bead-Beater®, and thecells lysed by rapid mixing for 6 cycles of 30 sec each. The homogenatewas centrifuged for 22 minutes in a Beckman J2-21M centrifuge using aJA-10 rotor at 9,000 rpm and 4° C. The supernatant was recovered andcentrifuged in a Beckman XL-80 ultracentrifuge using a TI45 rotor at36,000 rpm for 45 minutes at 4° C. The supernatant was recovered andPDE4D was precipitated by the addition of solid ammonium sulfate (0.33g/mL supernatant) while stirring in an ice bath and maintaining the pHbetween 7.0 and 7.5. Thirty minutes later, this mixture was centrifugedfor 22 minutes in a Beckman J2 centrifuge using a JA-10 rotor at 9,000rpm (12,000×g). The supernatant was discarded and the pellet wasdissolved in 100 mL of Buffer A (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5 mMMgCl₂, 14.2 mM 2-mercaptoethanol, 100 mM benzamidine HCl, and 5 μg/mLeach of leupeptin, pepstatin, aprotinin, calpain inhibitor I and II).The pH and conductivity were corrected to 7.5 and 15-20 mS,respectively.

At a flow rate of 0.67 mL/min, the resuspended sample was loaded onto a1.6×20 cm column (10 mL) of Sigma Cibacron Blue Agarose-type 300equilibrated in Buffer A. The column was washed in succession with 50 to100 mL of Buffer A, 20 to 30 mL of Buffer A containing 20 mM 5′-AMP, 50to 100 mL of Buffer A containing 1.5 M NaCl, and then 10 to 20 mL ofBuffer C (50 mM Tris HCl pH 8, 10 μM ZnSO₄, 14.2 mM 2-mercaptoethanol, 2mM benzamidine HCl). The enzyme was eluted with 20 to 30 mL of Buffer Ccontaining 20 mM cAMP.

The PDE4D activity peak was pooled and precipitated with ammoniumsulfate (0.4 g/mL enzyme pool) to remove excess cyclic nucleotide. Theprecipitated proteins were resuspended in Buffer X (25 mM MOPS pH 7.2, 5μM ZnSO₄, 50 mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desaltedvia gel filtration on a Pharmacia PD-10® column per manufacturer'sinstructions. The enzyme pool was dialyzed overnight against Buffer Xcontaining 50% glycerol. This enzyme preparation was quick-frozen in adry ice/ethanol bath and stored at −70° C.

The resultant preparations were about >80% pure by SDS-PAGE. Thesepreparations had specific activities of about 20 to 50 μmol cAMPhydrolyzed per minute per milligram protein.

Purification of PDE5 from S. cerevisiae

Cell pellets (29 g) were thawed on ice with an equal volume of LysisBuffer (25 mM Tris HCl, pH 8, 5 mM MgCl₂, 0.25 mM DTT, 1 mM benzamidine,and 10 μM ZnSO₄). Cells were lysed in a Micro-fluidizer® (MicrofluidicsCorp.) using nitrogen at 20,000 psi. The lysate was centrifuged andfiltered through 0.45 μm disposable filters. The filtrate was applied toa 150 mL column of Q SEPHAROSE® Fast-Flow (Pharmacia). The column waswashed with 1.5 volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄) and eluted with a step gradient of125 mM NaCl in Buffer A followed by a linear gradient of 125-1000 mMNaCl in Buffer A. Active fractions from the linear gradient were appliedto a 180 mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane(pH 6.8), 1 mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄, and 250 mM KCl). Afterloading, the column was washed with 2 volumes of Buffer B and elutedwith a linear gradient of 0-125 mM potassium phosphate in Buffer B.Active fractions were pooled, precipitated with 60% ammonium sulfate,and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mMNaCl, 0.5 mM DTT, and 10 μM ZnSO₄) The pool was applied to a 140 mLcolumn of SEPHACRYL® S-300 HR and eluted with Buffer C. Active fractionswere diluted to 50% glycerol and stored at −20° C.

The resultant preparations were about 85% pure by SDS-PAGE. Thesepreparations had specific activities of about 3 μmol cGMP hydrolyzed perminute per milligram protein.

Preparation of PDE7 from S. cerevisiae

Cell pellets (126 g) were thawed and resuspended at room temperature forabout 30 minutes with an equal volume of Lysis Buffer (50 mM Tris HCl,pH 8, 1 mM EDTA, 1 mM DTT, 50 mM NaCl, 2 mM benzamidine HCl, and 5 μg/mLeach of pepstatin, leupeptin, and aprotinin). The cells were lysed at0-4° C. with the aid of glass beads (125 mL) in a Bead-Beater® for 6×30second cycles. The lysate was centrifuged and filtered through 0.45 μmdisposable filters. The filtered extract (178 mL) was distributed into 4mL aliquots, quick-frozen with dry ice, and stored in a freezer at −70°C. These preparations were stable to several cycles of freezing andthawing and had specific activities of about 50 to 100 μmol cAMPhydrolyzed per minute per milligram protein.

Lipopolysaccharide-Stimulated TNFα Release from Human Peripheral BloodLymphocytes

To assess the ability of a compound to reduce TNFα secretion in humanperipheral blood lymphocytes (PBL), the following tests were performed.Previous studies have demonstrated that incubation of human PBL withcAMP-elevating agents, such as prostaglandin E21 forskolin,8-bromo-cAMP, or dibutryl-cAMP, inhibits the secretion of TNFα by thecells when stimulated by lipopolysaccharide (LPS; endotoxin).Accordingly, preliminary experiments have been performed to demonstratethat selective PDE4 inhibitors, such as rolipram, inhibit LPS-inducedTNFα secretion from human lymphocytes in a dose-dependent fashion.Hence, TNFα secretion from human PBL was used as a standard for theability of a compound to elevate intracellular cAMP concentrationsand/or to inhibit PDE4 activity within the cell.

Heparinized blood (approximately 30 mL) drawn from human volunteers wasmixed 1:1 with Dulbecco's modified phosphate-buffered saline. Thismixture was mixed 1:1 with HISTOPAQUE and centrifuged at 1,500 rpm atroom temperature without braking in the swinging bucket of a Beckmanmodel TJ6 centrifuge. Erythrocytes were centrifuged to the bottom of thetubes, and serum remained at the surface of the tubes. A layercontaining lymphocytes sedimented between the serum and HISTOPAQUE®layers, and was removed by aspiration to a fresh tube. The cells werequantified and adjusted to 3×106 cells/mL and a 100 μL aliquot is placedinto the wells of a 96 well plate. Test compounds and RPMI media(Gibco/BRL Life Sciences) are added to each of the wells 15 minutesprior to addition of bacterial LPS (25 mg/mL). The mixture was allowedto incubate for 20 hours at 37° C. in a humidified chamber. The cellsthen were separated by centrifuging at 800 rpm for 5 minutes at roomtemperature. An aliquot of 80 L of supernatant was transferred to a newplate or determination of TNFα concentration. TNFα protein in the cellsupernatant fluids was measured using a commercially availableenzyme-linked immunosorbent assay (ELISA) (CYTOSCREEN® Immunoassay Kitfrom Biosource International).

The cell-based assay provided the following results for variouscompounds of the present invention. The EC₅₀ values (i.e., effectiveconcentration of the compound capable of inhibiting 50% of the totalTNFα ) illustrate the ability of the present compounds to inhibitLPS-stimulated TNFα release from human PBL.

The following summarizes the IC₅₀ values determined for compounds ofstructural formulae (I) and (II) against human recombinant PDE4. In thefollowing table, R³ is hydrogen and n is 1, unless otherwise noted.

Example R¹ R² PDE4 IC₅₀ (μM)

(I) Et = ethyl H = hydrogen 22

Et 0.15 C₆H₄NO₂ 3

Et 0.20 C₆H₄Cl 8

Et 0.59 C₈H₇O 18

Et 0.62 C₆H₄F 20

Et 0.99 C₆H₁₁ 19

Et 1.28 C₃H₄N 38

Et 1.30 C₆H₆N 33

Et 1.40 C₆H₄NO₂ 41

Et 1.42 C₆H₆N 34

Et 1.65 C₆H₄NO₂ 28

Et 2.28 C₇H₄F₃ 26

Et 3.07 C₆H₄Br 10

Et 4.38 C₇H₇O₂S 11

Et 5.45 C₇H₆NO 35

Et 5.74 C₅H₃N₂O₂ 5

Et 6.10 C₇H₇ 1

Et 8.22 C₈H₁₀N 30

Et 13.42 C₇H₅N₂O₄ 44

H 54.16 C₆H₅

(II) Et = ethyl H = hydrogen 13

Et 0.15 C₈H₁₀N 39

Et 0.20 C₆H₄NO₂ 12

Et 0.22 C₇H₆NO 21

Et 0.59 C₆H₅ 40

Et 1.29 C₆H₆N 42

Et 1.49 C₆H₃Cl₂ 4

Et 2.90 C₇H₇ 9

Et 4.58 C₇H₇O₂S 2

Et 5.45 C₆H₄Cl 29

Et 6.78 C₇H₄F₃ 7

Et 6.83 C₈H₇O 31

Et 5.13 C₇H₆NO₂ 6

Et 8.55 C₆H₄Br 46

Et 8.70 C₉H₉O₂ 17

Et 13.21 C₆H₄F 32

Et 13.39 C₅H₄N 43

H 15.63 C₆H₄NO₂ 37

Et 16.00 C₆H₆N 24

Et 16.53 C₇H₅O₂ 21

Et 31.29 C₆H₅ 36

Et 110.00 C₆H₄NO₂ 23

Et 133.01 C₅H₃N₂O₂ 45

Et 193.71 C₈H₃F₆

The data presented above shows that compounds of formula (I) and (II)are potent and selective inhibitors of PDE4, e.g., the compounds have anIC₅₀ vs. human recombinant PDE4 of about 0.15 to about 195 μm. As anadded advantage, the compounds of formula (I) and (II) reduced oreliminated the adverse side effects, such as CNS effects, associatedwith prior PDE4 inhibitors. In particular, compounds of formulae (I) and(II) were tested in cell-based assays and in animal models to illustratethe efficacy of the compounds with respect to inhibiting PDE4 both invitro and in vivo.

The cell-based assay provided the following results for various pyrrolecompounds of the present invention. The EC₅₀ values (i.e., effectiveconcentration of the compound capable of inhibiting 50% of the totalTNFα) illustrate the ability of the present compounds to inhibitLPS-stimulated TNFα release from human peripheral blood lymphocytes.

PDE4 IC₅₀ PBL/TNFα EC₅₀ Compound (μm) (μm) Ex. 22 .15 .87 Ex. 3  .201.50 Ex. 21 .40 10.00 Ex. 40 1.29  6.72 Ex. 42 1.49  100.0

The above Table and FIG. 1 illustrate the ability of compounds offormulae (I) and (II) to inhibit PDE4 activity and TNFα release.Preferred compounds have a PBL/TNFα EC₅₀ about 500 nM or less, andpreferably about 200 nM or less. More preferred compounds have aPBL/TNFα EC₅₀ of about 100 nM or less.

To achieve the full advantages of the present invention, the compoundhas an IC₅₀ vs. human recombinant PDE4 of about 100 nM or less and aPBL/TNFα EC₅₀ of about 500 nM or less. More preferably, the compound hasan IC₅₀ of about 50 nM or less and a PBL/TNFα EC₅₀ of about 100 nM orless.

Animal Models

Assay for Inhibition of Serum TNFα Levels in Mammals (Mouse/TNFα ED₅₀(mg/kg))

In order to assess the ability of a compound to reduce serum TNFα levelsin mammals, the following protocol was employed. Those skilled in theart appreciate that previous studies have demonstrated that incubationof LPS-activated human monocytes with agents that can elevate cAMP, likePGE2, forskolin, and the dbcAMP, inhibited secretion of TNFα. PDE4inhibitors like rolipram, which also elevate cAMP, have been found toinhibit serum TNFα as well. Rolipram has also been found to inhibitsecretion of TNFα from LPS-activated mouse macrophages. Accordingly, invivo efficacy of a PDE4 reducing compound was shown by dosing withcompound and measuring reduction of serum TNFα levels in LPS-injectedmice. Female C3H mice, 20-25 m body weight, were fasted overnight anddosed intraperitoneally with test compound in appropriate vehicle 60minutes before LPS injection. Five μg of LPS was then injectedintraperitoneally into the mice. Ninety minutes after LPS injection,mice were bled from the heart. Blood was allowed to clot overnight at 4°C. Samples were centrifuged for 10 minutes in a microcentrifuge and theserum removed and stored at −20° C. until analysis. Serum levels of TNFαwere subsequently measured using a commercially available ELISA kit(Genzyme) following the protocol enclosed in the kit. The inhibition ofserum TNFα levels caused by the compound was determined relative toserum TNFα levels in control mice receiving vehicle alone. The resultsare summarized in the plots of FIG. 1.

Combined Mouse endotoxin-stimulated TNFα Release and Locomotor ActivityAssay

The purpose of this study was to determine the efficacy of PDE4inhibitors in vivo in an LPS mouse model together with a determinationwith respect to central nervous system (CNS) side-effects manifested bya decrease in spontaneous mobility.

The test animals were female Balb/c mice, having an average weight ofabout 20 g. The PDE4 inhibitors, formulated in 30% Cremophor® EL, wereadministered via intraperitoneal (i.p.) injections at doses of 0.1, 1.0,10.0, and 100 mg/kg. Individual dose volumes (about 150 L) were adjustedbased on the body weights measured. One hour later, 5 mg/kg LPS in afinal volume of 200 μL was injected via the tail vein to each animal.Ninety minutes following the LPS treatment, the animals were bled andserum samples were collected before being stored at −70° C. untilassayed.

For efficacy determination, the serum samples were diluted two-fold andTNFα levels were determined using the CYTOSCREEN® Immunoassay Kit(Biosource International) The data were averaged between triplicatesample subjects for each of the tested compounds.

For side-effect profiling, a subjective visual scoring system wasutilized at 5 min and 20min after administration of PDE4 inhibitors.Vehicle control animals were rated a single “+” and animals that wereeffectively immobilized and stretched out on the bottom of the cage withlittle detectable movement were rated as “++++.” Alternatively, asemi-automated “open field” system (e.g., a Photo-beam ActivityMeasurement System as sold by San Diego Instruments) for monitoringmovements was used for assessing the effect of PDE4 inhibitors on miceand/or rats. In this instance, the subjects could be monitoredcontinuously over pre-set intervals.

Movement of the X-Y plane, or rearing up on the hind legs, wasquantified by counting the number of “light-beam” crosses per unit oftime. A decrease in the number of activity events is directlyproportional to the mobility or immobilization of the animal. Thequantitative scoring correlated well with the subjective measurementsdescribed above.

% 1 10 100 Seda- TNFα Vehicle mg/ mg/ mg/ Doses tive In- Compoundcontrol kg kg kg (mg/kg) Effect hibition Comparative 3 3 3  1 −−  43Example¹⁾  10 + 100 100 +++ 100 Example 22 3 3 3 3  1 −−  13  10 −−  84100 −− 100 ¹⁾trans-3-(3-cyclopentoxy-4-methoxyphenyl)-1-methoxycarbonyl-4-methyl-4-(methylcarbonyl)pyrrolidine, i.e., Example 12 of Feldman et al.U.S. Pat. No. 5,665,754, incorporated herein by reference.

The data presented above show that compounds of formulae (I) and (II)are potent and selective inhibitors of PDE4. As an important addedadvantage, the compounds of formulae (I) and (II) also reduced oreliminated the adverse CNS side effects associated with prior PDE4inhibitors. Compounds of formulae (I) and (II) were further tested foremetogenic properties in animal models to further illustrate theefficacy of the compounds. The method and results of the emetogenic testare set forth below.

The compounds of the present invention are useful for selectivelyinhibiting PDE4 activity in a mammal, without exhibiting the adverse CNSand emetic effects associated with prior PDE4 inhibitors.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and, therefore, only such limitations should be imposed asare indicated by the appended claims.

What is claimed is:
 1. A method of treating a mammal having a conditionwhere inhibition of a cAMP-specific PDE is of therapeutic benefit, saidmethod comprising administering to said mammal a therapeuticallyeffective amount of a compound having a formula

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)-alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl;R⁴ and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3.
 2. A method of modulating cAMP levels in a mammal comprisingadministering to said mammal an effective amount of a compound of

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl; R⁴and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3.
 3. A method of treating a mammal having a condition where inhibitionof a cAMP-specific PDE is of a therapeutic benefit comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising a compound having a formula

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl; R⁴and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3; a pharmaceutically acceptable carrier; and, optionally, a secondanti-inflammatory therapeutic agent.
 4. The method of claim 3 whereinthe condition is an allergic disease, an autoimmune disease, aninflammatory disease, an arthritic disease, or dermititis.
 5. The methodof claim 3 wherein the condition is rheumatoid arthritis,osteoarthritis, gouty arthritis, or spondylitis.
 6. The method of claim3 wherein the condition is thyroid-associated ophthalmopathy, Behcetdisease, sepsis, septic shock, endotoxic shock, gram negative sepsis,gram positive sepsis, toxic shock syndrome, allergic conjunctivitis,vernal conjunctivitis, or eosinophilic granuloma.
 7. The method of claim3 wherein the condition is asthma, chronic bronchitis, allergicrhinitis, adult respiratory distress syndrome, chronic pulmonaryinflammatory disease, chronic obstructive pulmonary disease, silicosis,or pulmonary sarcoidosis.
 8. The method of claim 3 wherein the conditionis reperfusion injury of the myocardium, brain or extremities as a brainor spinal cord injury due to minor trauma.
 9. The method of claim 3wherein the condition is a fibrosis, keloid formation, or scar tissueformation.
 10. The method of claim 3 wherein the condition is systemiclupus erythematosus, a transplant rejection disorder, a graft vs. hostreaction, or an allograft rejection.
 11. The method of claim 3 whereinthe condition is chronic glomerulonephritis, an inflammatory boweldisease, Crohn's disease, or ulcerative colitis.
 12. The method of claim3 wherein the condition is proliferative lymphocytic disease or aleukemia.
 13. The method of claim 3 wherein the condition is aninflammatory dermatosis, atopic dermatitis, psoriasis, or urticaria. 14.The method of claim 3 wherein the condition is a cardiomyopathy,congestive heart failure, atherosclerosis, pyrexia, cachexia, cachexiasecondary to infection of malignancy, cachexia secondary to acquiredimmune deficiency syndrome, ARC, cerebral malaria, osteoporosis, a boneresorption disease, fever and myalgias due to infection, diabetesinsipidus, a central nervous system disorder, depression, multiinfarctdementia, an anxiety or stress response, cerebral ischemia, tardivedyskinesia, Parkinson disease, and premenstrual syndrome.
 15. The methodof claim 3 wherein the mammal exhibits a minimal emetic response. 16.The method of claim 3 wherein the mammal is free of an emetic response.17. The method of claim 3 wherein the mammal exhibits minimal adversecentral nervous system side effects.
 18. The method of claim 3 whereinthe mammal is free of adverse central nervous system side effects. 19.The method of claim 3 wherein the second anti-inflammatory therapeuticagent is capable of targeting TNFα.
 20. A method of reducing TNF levelsin a mammal comprising administering to said mammal a therapeuticallyeffective amount of a compound having a formula

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl; R⁴and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3.
 21. A method of suppressing inflammatory cell activation in a mammalcaused by a pathway wherein cyclic AMP-specific phosphodiesterase isinvolved comprising administering to said mammal a therapeuticallyeffective amount of a compound having a formula

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl; R⁴and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3.
 22. A method of inhibiting PDE4 function in a mammal comprisingadministering to said mammal a therapeutically effective amount of acompound

wherein R¹ is nitro-substituted phenyl; R² is alkyl or hydrogen; R³ isselected from the group consisting of alkyl, alkoxy, alkoxyalkyl,aralkoxyalkyl, C(═O)alkyl, NR⁴R⁵, C(═O)NR⁵R⁶, C(═O)Oalkyl, CO₂H,OC(═O)alkyl, nitro, halo, alkylthio, SO₂(alkyl), SO₃H, and haloalkyl; R⁴and R⁵, independently, are hydrogen or alkyl, or R⁵ or R⁶ are takentogether to form a 5- or 6-membered carbocyclic ring; and n is 0 through3.
 23. The method of claim 1 wherein R¹ is selected from the groupconsisting of:


24. The method of claim 1 wherein R² is selected from the groupconsisting of hydrogen and C₁-C₆ alkyl.
 25. The method of claim 1wherein R² is hydrogen or C₁₋₂alkyl, and n is
 0. 26. The method of claim1 wherein the compound is selected from the group consisting of:2-methyl-1-(3-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylic acid ethylester; 2-methyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylic acidethyl ester; 2-methyl-1-(2-nitrophenyl)-5-phenyl-1H-pyrrole-3-carboxylicacid ethyl ester;5-methyl-1-(4-nitrophenyl)-2-phenyl-1H-pyrrole-3-carboxylic acid ethylester; 5-methyl-1-(3-nitrophenyl)-2-phenyl-1H-pyrrole-3-carboxylic acid;and 1-(3-nitrophenyl)-5-methyl-2-phenyl-1H-pyrrole-3-carboxylic acidethyl ester.